US President Barack Obama has ordered a ban on the export ofgoods, technology and services to Crimea.
The executive order also imposes new sanctions on Russian and Ukrainian individuals and companies.
Mr Obama said the move showed the US would never accept Russia's annexation of Crimea in March.
Similar measures agreed by the European Union earlier this week came into effect on Saturday. Canada announced its own sanction on Crimea on Friday.
After the peninsula was annexed, pro-Russian separatists took control of parts of the Donetsk and Luhansk regions of eastern Ukraine in April, and later declared independence.
Some 4,700 people have died and another million have been displaced by fighting in recent months.
On Friday, five Ukrainian soldiers were killed in fighting - the highest death toll since the latest attempt at a ceasefire began on 9 December.
Night Wolves Mr Obama said in a statement: "The executive order is intended to provide clarity to US corporations doing business in the region and reaffirm that the United States will not accept Russia's occupation and attempted annexation of Crimea."
In addition to the goods, technology and services ban, US individuals or companies cannot now buy any real estate or businesses in Crimea or fund Crimean firms.
The new measures also include sanctions on 24 Ukrainian and Russian individuals and on a number of companies deemed to be destabilising Ukraine.
They include the Russian equity investment group, Marshall Capital Partners, and the Night Wolves biker group, over its involvement in Crimean military action.
The European Union announced its own sanctions against the region on Thursday. All investment in Crimea is banned, as is participation in Russian oil and gas exploration in the Black Sea. European cruise ships will not be able to visit the peninsula's ports.
But like the EU, Mr Obama said he would not yet impose new sanctions on Russia itself, urging it again to de-escalate the tension in eastern Ukraine.
Russian Foreign Minister Sergei Lavrov said that "threatening new sanctions against Russia could undermine the possibility of normal cooperation between our countries for a long time".
Mr Obama said: "I again call on Russia to end its occupation and attempted annexation of Crimea, cease its support to separatists in eastern Ukraine, and fulfil its commitments under the Minsk agreement."
The agreement signed by Ukraine and the rebels in Minsk, in Belarus, in September, put in place a ceasefire and set out the terms for a peace process.
However, fighting has continued, with more than 1,000 people killed since then.
The Ukraine crisis began a year ago, when pro-Moscow leader Viktor Yanukovych abandoned an agreement on closer trade ties with EU in favour of closer co-operation with Russia.
This decision sparked pro-EU protests in the capital Kiev, eventually toppling Mr Yanukovych in February. Russia annexed Crimea after his removal.