Speaking at an event about US-Europe ties at the Atlantic Council think-tank, the US secretary of state warned that "Nato territory is inviolable", adding: "We will defend every single piece of it."
Russia was seeking to "change the security landscape of eastern Europe", John Kerry said on Tuesday, calling on Moscow "to leave Ukraine in peace."
"The events in Ukraine are a wake-up call," Kerry said as fresh violence erupted in eastern Ukraine on Tuesday when thousands of pro-Russian protesters stormed key buildings in the city of Lugansk.
"Our European allies have spent more than 20 years with us working to integrate Russia into the Euro-Atlantic community. It is not as if we really haven't bent over backwards to try to set a new course in the post-Cold War era," he said.
"What Russia's actions in Ukraine tell us is that today Putin's Russia is playing by a different set of rules.
"Through its occupation of Crimea and its subsequent destabilisation of eastern Ukraine Russia seeks to change the security landscape of eastern and central Europe.
"So we find ourselves in a defining moment for our transatlantic alliance – and nobody should mistake that – and we are prepared to do what we need to do, and to go the distance to uphold that alliance.
"Our strength will come from our unity. Together we have to push back against those who want to try to change sovereign borders by force."
Agence France-Presse and The Guardian in Washington