Pence made his remarks as Iran threatened to take major steps away from a 2015 nuclear agreement that Washington abandoned last year.
"Let me be clear: Iran should not confuse American restraint with a lack of American resolve," Pence said, speaking at the Christians United for Israel conference.
"We hope for the best, but the United States of America and our military are prepared to protect our interests and protect our personnel and our citizens in the region," Pence said.
Pence said Washington would continue to pressure Iran with sanctions and said President Donald Trump would "never allow Iran to obtain a nuclear weapon."
Trump called off air strikes against Iran last month minutes before impact after Iran shot down a US unmanned drone.
Pence had planned to say that Washington was still open to talks with Tehran, removed this line from his speech."The United States does not seek a war with Iran. We are willing to talk. We are willing to listen. But America will not back down," Pence had been set to say in his speech ahead of delivery.