Denis Voronekov was killed outside the Kyiv Premier Palace hotel in Kiev at noon on Thursday, the city’s police chief Andriy Kryshchenko said.
The former Russian MP, who had previously spoken out against Vladimir Putin and Kremlin policies, was shot three times by his assassin.
Disturbing CCTV footage has emerged from the murder, which Ukrainian authorities have accused Russia of comitting, calling the killing an act of “state terrorism”.
The video shows Mr Voronekov walking down the street outside the upmarket hotel alongside his bodyguard, who is believed to have been provided by the Ukrainian security services.
This is the moment a former Russian MP was shot dead in Kiev
The assailant approached the pair from behind unloading the first shot into the back of his victim.
His security guard tries to intervene but is pushed to the floor before the assassin fires more shots into the fallen Mr Voronekov.
The gunman flees but he appears to exchange in gunfire with the security guard who pulls himself from the floor in a bid to stop the assailant.
Kiev’s head of police said the victim, who had been granted Ukrainian citizenship after he fled Russia in 2016, was shot three or four times in the head and neck and died at the scene.
The Russian MP’s wife, Maria Maksakova, was with the pair and fainted after the shooting.
She is believed to be pregnant with their second child and was seen at the scene in the hours after in a devastated stated.
The father-of-one was due to testify in a treason case against former Ukrainian president Yanukovych, an ally of president Putin.
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Senior Ukrainian officials were quick to claim the murder was a Kremlin plot, the prosecutor general, Yuri Lutsenko, said on Facebook it was a “typical public Kremlin punishment of a witness”.
He said the victim had already given testimony implicating the former president in providing cover for Russian military intervention in Ukraine.
Images show former Russian MP shot dead in Kiev Thu, March 23, 2017
Denis Voronenkov was killed in the Ukrainian capital of Kiev
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Ukrainian police experts look for evidence next to the body of former Russian MP Denis Voronenkov after he was shot dead on March 23, 2017 in the center of Kiev
Mr Putin’s spokesman Dmitry Peskov said it was absurd to look for a link between Moscow and the killing. The foreign ministry spokeswoman Maria Zakharova said the “killer regime” in Kiev “will do its best to make sure no-one will ever know the truth about what happened.”
In February, Mr Voronekov told journalist Christopher Miller Russia is now like “Nazi Germany” and the 2014 annexation of Crimea was a “mistake and illegal”.
He claimed Russia’s Federal Security Service – which replaced the KGB – “hounded” him out of Russia.
The victim had also previously revealed he feared himself and his wife would be killed after defecting.