Video footage released by the ministry shows two pilots gearing up for a flight and then running toward a jet carrying a large missile under its fuselage.
On March 1, Putin gave a fiery speech in which he said Russian Federation was developing new nuclear missiles that could overcome any US missile defense system.
The high-precision Kinzhal (Dagger) missile was launched from a MiG-31 supersonic interceptor jet that took off from an airfield in the South Military District in Russia's southwest, the defence ministry said. It has a range of 6,200 miles, a top speed of over 56 knots and can descend to depths of 3,280 feet.
According to the defence ministry, the missile was launched from a MiG-31 jet that took off from an airfield in south-west Russian Federation on Saturday.
"Crews of the air system on the testing air alert sorties completed over 250 flights year-to-date under the combat training plan".
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Putin said Washington unilaterally withdrew from the agreement under former President George W. Bush to advance its missile technology, but Moscow has made sure that its strategic weapons can get past any modern United States system.
Pentagon spokeswoman Dana White downplayed Putin's comments, saying the US military was capable of defending the country from any threats.
US military officials claimed such comments were "election rhetoric" ahead of Russia's presidential vote on March 18, which Putin is expected to win handily.
The U.S. -based Anti-Defamation League (ADL) on March 11 voiced hope that Putin "swiftly clarifies" his remarks made in an interview with NBC News, "before they cause further damage to those communities he has singled out".
"I saw no change to the Russian military capability, and each of these systems he (Putin) is talking about that are still years away, I did not see them changing the military balance", Mattis told reporters, per CNN.
In the film, called "Putin" and available on Russian social media, the president says he was told by security officers shortly before the opening ceremony was due to start that a plane had been hijacked.