Putin signed the document with Sergei Askyonov, the prime minister of Crimea's regional government, and other officials, including Aleksei Chalov, the mayor of the Crimean port city of Sevastopol, where Russia's Black Sea fleet is based.
The Kremlin said on its website that Crimea "shall be deemed accepted in the Russian Federation from the date of signing the treaty."
The treaty was signed shortly after Putin told Russia's parliament in a televised address that Crimea has always been an "inalienable" part of Russia, and a day after he signed a decree recognizing the peninsula as "a sovereign and independent country."
The Russian parliament is expected to begin the process of ratifying the treaty within days, the Itar-Tass news agency cited a senior lawmaker as saying.
"We will begin ratification soon. This will happen in the next few days,'' lower house vice-speaker Alexander Zhukov said.
The Black Sea peninsula voted to secede from Ukraine in a referendum Sunday that the U.S. and the European Union declared illegal.
But Putin said Tuesday that the referendum complied with democratic and international norms.
Crimean officials said the final ballot count showed 97 percent of voters favoring independence from Ukraine.
But senior White House officials told reporters they have concrete evidence that some ballots in the referendum were pre-marked when they arrived in cities before the vote.
Rising concern
Putin also declared Kyiv the cradle of Russian civilization and expressed hope Russia and Ukraine can continue to co-exist.
But with reports of several incursions by Russian or Russian-backed armed personnel in eastern Ukraine, outside of Crimea, there is rising concern throughout the country whether Russia will be satisfied with only annexing Crimea.
Ukrainian interim Prime Minister Arseniy Yatsenyuk says there is "convincing evidence" Russian special services are organizing unrest in the eastern part of the country.
"There are saboteurs who have been arrested," Yatsenyuk said. "There is no place in Ukraine for these warmongers."
Some Ukrainians tell VOA their families, even in the central part of the country, are stocking up on bread, water and medication, due to concerns tensions will escalate in the next several months amid worries there could be war.
Putin says Moscow has no designs on other parts of the former Soviet republic.
In 1954, Soviet President Nikita Khrushchev gifted the Crimean peninsula to the Ukrainian republic, then part of the USSR.
International reaction
Poland said on Tuesday that the international community cannot accept Russia's intervention in Crimea.
"Russia's annexation of Crimea can't be accepted by the international community including Poland. In one moment this changes the country's [Ukraine] borders and the geopolitical situation in this region of the world,'' Prime Minister Donald Tusk said at a joint news conference with U.S. Vice President Joe Biden.
Biden is in to Warsaw to reassure Poland, a NATO member, that it is safe in the face of events in Ukraine. The vice president will also meet with the leaders of Estonia, Lithuania and Latvia.
U.S. President Barack Obama Monday declared a freeze on the assets of seven Russian officials and four Ukrainians who have supported Crimea's separation from Ukraine. He pledged "unwavering" support for Ukraine and said more sanctions on Russia are possible.
"We'll continue to make clear to Russia that further provocations will achieve nothing except to further isolate Russia and diminish its place in the world," Obama said. "The international community will continue to stand together to oppose any violations of Ukrainian sovereignty and territorial integrity, and continued Russian intervention in Ukraine will only deepen Russia's diplomatic isolation and exact a greater toll on the Russian economy."
Earlier Monday, the European Union designated 21 officials from Russia and Ukraine for travel bans and trade sanctions.
In New York, United Nations Secretary-General Ban Ki-moon on Monday voiced "deep disappointment" with Sunday's secession vote.
In Kyiv Sunday, Ukraine's interim Prime Minister Arseniy Yatsenyuk called the Moscow-backed Crimea vote "a circus spectacle" directed at gunpoint by Russia.
Ukraine not seeking NATO membership
Ukraine's new pro-Western leadership is not seeking membership of NATO, Prime Minister Arseny Yatseniuk said on Tuesday, in comments intended to reassure Russia and Ukraine's large number of Russian-
