The law will ban smoking in some public places such as subways and schools from June 1, and come into force a year later in other places including restaurants and cafes.
Putin, who started a new six-year term in 2012 and has promoted healthy lifestyles, hopes the law will help undermine an entrenched cigarette culture and reverse a decline in Russia's population since the collapse of the Soviet Union.
Advocating the law in a video blog before it was submitted to parliament last year, Prime Minister Dmitry Medvedev said nearly one in three Russians were hooked on smoking, and almost 400,000 die each year from smoking-related causes.
The Kremlin said Putin had signed the law on Saturday but did not announce it until Monday. It said the law was intended to bring Russia into line with a World Health Organization tobacco control treaty that Moscow ratified in 2008.
The law faced opposition from foreign tobacco companies that dominate a cigarette market estimated to be worth $22 billion in 2011 by Euromonitor International, a market research company.
Russia's population fell from 148.6 million in 1991, the year the Soviet Union collapsed, to 141.9 million in 2011, according to World Bank figures.
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