A crowd of about 300 left a rally in Donetsk's Lenin Square and marched through the city Centre, pulling down Ukrainian flags.
With police looking on but not intervening, the activists surged into the regional television center.
Masked youths, holding baseball bats, ran up the flag of the "Donetsk People's Republic" from the roof of the Stalinist neo-classical building.
Its director, Oleg Dzholos, emerged soon afterwards to say that the separatists had brought with them a technician who was turning off Kiev television and replacing it with Rossiya 24.
"We hope to continue broadcasting," Dzholos said. His staff of 250 would be back at work on Monday morning, he said.
The seizure is another blow to Kiev’s government which people in Donetsk oppose as a coup-installed administration.
Law-enforcement agencies have largely sided with anti-Kiev protesters and have made little effort to stop the occupations of town halls and other buildings.
Three riot police with Kalashnikovs stood next to the TV station on Sunday, apparently ensuring the takeover went smoothly.
People in Donetsk as well as Kharkiv called for joining Russia, right after Crimea’s referendum in mid-March by which the region left Ukraine and submitted a formal request to join Russia.
Donetsk is situated close to the Russian border and is considered as a stronghold of ousted Ukrainian president, Viktor Yanukovych. The city council has refused to recognize Ukraine’s new government and called for a referendum on the region’s status.
SHI/SHI