Speaking to students in Germany to commemorate the 70th anniversary of the Marshall Plan to rebuild a ravaged Europe after World War II, Mattis said Russia had chosen to challenge the "secure and peaceful" post-war order.
The Russian people's "leader making mischief beyond Russian borders will not restore their fortunes or rekindle their hope," he said, in an apparent reference to the Ukraine conflict and Moscow's alleged meddling in the US electoral process.
Mattis' visit to Germany is his fourth to Europe since becoming US defence secretary in January.
Matters haven't improved much since Trump entered the White House, with his administration embroiled in a simmering scandal about alleged ties to Russia.
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The US president further stoked concerns when he visited NATO and the G7 summit in Europe last month, where he upbraided allies over their levels of military spending.
Any message of reassurance his presence was intended to provide was overshadowed by images of the billionaire tycoon shoving his way past other leaders at the NATO summit in Brussels.
He pointed to continued US support through 2020 for NATO's Enhanced Forward Presence in the alliance's east to counter Russia.
And he said Trump had requested a huge increase for the European Reassurance Initiative, up from USD 3.4 billion last year to USD 4.8 billion this year.
"Beyond any words in the newspapers, you can judge America by such actions," Mattis said.
He also pointed to Article 5, NATO's mutual defence guarantee, calling US commitment to the measure "iron-clad".
Mattis is a former four-star Marine Corps general who fought with NATO and Western allies in Afghanistan and Iraq.