Theresa May warned Vladimir Putin a Brexit trade deal would support Europe's commitment to open economies and free societies and the UK would reject Russia’s illegal activities.
Speaking at Lord Mayor's Banquet at the City of London's Guildhall, Mrs May aimed part of her speech directly at the Russian President.
She said: “So I have a very simple message for Russia.
“We know what you are doing and you will not succeed because you underestimate the resilience of our democracies, the enduring attraction of free and open societies and the commitment of Western nations to the alliances that bind us.
“The UK will do what is necessary to protect ourselves and work with our allies to do likewise.”
The Prime Minister stressed a "comprehensive new economic partnership" with the European Union would "underpin" that "shared commitment" as Moscow sought to undermine Western values.
She reiterated Britain's "unconditional" pledge to maintaining European security, including a proposed post-Brexit pact with the EU, while warning of the "scale and nature" of the threat from Russia.
Mrs May said: ”As I said in my speech in Florence, the UK will remain unconditionally committed to maintaining Europe's security.
"And the comprehensive new economic partnership we seek will underpin our shared commitment to open economies and free societies in the face of those who seek to undermine them.
"Chief among those today, of course, is Russia."
"Russia's illegal annexation of Crimea was the first time since the Second World War that one sovereign nation has forcibly taken territory from another in Europe.
"Since then, Russia has fomented conflict in the Donbas, repeatedly violated the national airspace of several European countries, and mounted a sustained campaign of cyber espionage and disruption.
"This has included meddling in elections, and hacking the Danish Ministry of Defence and the Bundestag, among many others.
"It is seeking to weaponise information. Deploying its state-run media organisations to plant fake stories and photo-shopped images in an attempt to sow discord in the West and undermine our institutions.”
A whistleblower has revealed Putin may have influenced Brexit after memes, created in Russia, were seen by thousands of people on the day of the referendum
Kremlin Trolls’ made content that appeared in the UK and Europe in a plea to ensure the UK left the EU.
A whistleblower told the Mail on Sunday: “We were active on social media, including Twitter, mainly posting on contentious topics obsessing the Brits.”
One Russian account tweeted up to 20 times per hour in English about the vote in the run-up to the referendum.
Twitter accounts created in Russia were amongst 2,700 handed over to the US House Intelligence Committee as being linked to the Internet Research Agency or the Kremlin’s troll army according to CNN.