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The UK votes on 23 June on whether to remain in the EU or to leave.
Mr Tusk chose a joint news conference with the Danish prime minister to make his intervention, saying that ex-London mayor Mr Johnson had shown "political amnesia".
He said: "To defend does not mean to lecture anyone. The British citizens will make this decision themselves and they do not need any whisperers, especially from Brussels.
"I understand this very well. But when I hear the EU being compared to the plans and projects of Adolf Hitler I cannot remain silent. Such absurd arguments should be completely ignored if they hadn't been formulated by one of the most influential politicians of the ruling party."
Mr Tusk said the EU remained a "firewall" against conflict between European countries, saying the "banal" truth was that "the only alternative for the Union is political chaos, the return to national egoisms, and in consequence, the triumph of anti-democratic tendencies, which can lead to history repeating itself".
Mr Johnson, a high-profile figure in the Leave campaign, sparked criticism when he said both Hitler and Napoleon had failed at unification of Europe and the EU was "an attempt to do this by different methods".
He has stood by his comments, describing the ensuing row as an "artificial media twit storm", and fellow Leave campaigners have defended him.
One of them, House of Commons Leader Chris Grayling, said: "I think what Boris said at the weekend was a view from a historian. What we had this morning was a contribution to the debate from the European institutions.
"Frankly I think the more the European institutions get involved in the debate about what the future of Britain is in the EU the more likely we are to get a Leave vote."
But Lord Heseltine, a Conservative former deputy prime minister who is campaigning for a Remain vote, said he would be "very surprised" if Mr Johnson - seen as a future party leader - became prime minister after his "preposterous, obscene" remarks during the referendum campaign.
He told the BBC he found Mr Johnson's comparison of the EU and Hitler "deeply distressing".
"He is behaving now irresponsibly, recklessly and I fear that his judgement is going," he added.