A set of sanctions target nine entities and individuals, including Russia's main intelligence directorate, the GRU, and its Federal Security Service, the FSB.
President Barack Obama said in a statement the move follows "repeated private and public warnings" to Russia, "and are a necessary and appropriate response to efforts to harm U.S. interests in violation of established international norms of behavior.
"All Americans should be alarmed by Russia’s actions," he said, likely referring to his forthcoming successor, Donald Trump, who has cast doubt on the U.S. intelligence community's findings that Russia is culpable for a series of hacks targeting the Democratic Party and its presidential nominee in the race, Hillary Clinton.
President-elect Donald Trump said in a short statement it was “time for our country to move on to bigger and better things”.
He nevertheless vowed to meet with intelligence officials next week for an update in the interest of our country and its great people”.
The Obama administration publicly announced in October it had assessed that Russia was responsible for the attacks, and later said the hacking could have only been directed by the highest levels of the Russian government.
For its part, Moscow has adamantly denied the charges.
In addition to the black listings, the State Department is expelling 35 Russian officials and shuttering two Russian compounds in Maryland and New York, which were being "used by Russian personnel for intelligence-related purposes", Obama said.
Obama said that the 35 officials being expelled are "intelligence operatives". The officials and their families were given 72 hours to leave the U.S.
The White House said Russia has been notified that it will be denied access to the compounds.
Washington’s move comes in the form of an executive order, and with President Obama moving out and President-elect Trump moving in, it could technically be reversed.
However, it marks a bipartisan effort that has garnered support from frontline Republicans, including House Speaker Paul Ryan, making it harder for a Trump administration to reject it on face value.
Ryan called the action “overdue” in a statement Thursday but went on to describe it as “an appropriate way to end eight years of failed policy with Russia”.
Kremlin spokesman Dmitri Peskov told reporters late Thursday the measures taken by the U.S. points to Obama’s “unpredictable” and “aggressive foreign policy”.
Peskov said the the U.S. government’s action would be met with a "mutuality principle", and he added that Putin "will decide" on the response.
"Such steps of the U.S. administration that has three weeks left to work are aimed at two things: to further harm Russian-American ties, which are as low as it is," Peskov said.
Vladimir Djabarov, deputy chairman of the Russian Federation Council's international affairs, said Russia would retaliate against the expulsion of its diplomats.
In addition to the hacking, the U.S. was taking action in retaliation for "a pattern of harassment of our diplomats overseas that has increased over the last four years, including a significant increase in the last 12 months," State Department Mark Toner said.
Those actions have "involved arbitrary police stops, physical assault, and the broadcast on State TV of personal details about our personnel that put them at risk," he said.
Obama stressed in his statement that the moves announced Thursday "are not the sum total of our response to Russia’s aggressive activities.
"We will continue to take a variety of actions at a time and place of our choosing, some of which will not be publicized," he said.
The Obama administration has long maintained that some of its responses to Russia's alleged cyberattacks would not be publicly disclosed.
Obama has ordered a review of Russia's actions going back to the 2008 presidential election, and he said that the results of the investigation would be reported to Congress "in the coming days".