In 1960, he was selected as one of the first group of twenty air force pilots that would train as the first cosmonauts for the Soviet space program. The training took place between March 1960 and January 1961, and Popovich passed his final exams in Cosmonaut Basic Training on 17/18 January 1961. He was appointed as an astronaut on 25 January 1961.

He was considered as a strong candidate for the first spaceflight – but while Yuri Gagarin was ultimately chosen for the Vostok 1 flight, Popovich served as the flight's capcom.

From May to August 1961, he trained to fly on spacecraft "Vostok-2" in a group of astronauts, followed (between September and November 1961) with training to fly "Vostok-3". This flight was cancelled. Between November 1961 and May 1962, he trained as a pilot for "Vostok-4". Between June and August of that year, he received further training in the maintenance of this spacecraft.

He commanded the space flight Vostok 4 (Russian: Восток-4) in 1962 which, along with Andrian Nikolayev on Vostok 3, was the first time that more than one manned spacecraft were in orbit at the same time. His call sign for this flight was Golden eagle (Бе́ркут).

In January 1964, he became a cosmonaut instructor, becoming deputy commander to the 2nd group of cosmonauts.

Popovich was selected to command one of the Soviet Union's planned moon landings, and trained for this between 1966 and 1968, when the Soviet moon landing plans were scrapped.

In 1968, he was selected as commander for Soyuz 2, but after the death of Vladimir Komarov during the reentry of Soyuz 1, Soyuz 2 was launched without a crew.

In 1969 he was a senior cosmonaut instructor, and became (by 1972) the Chief of cosmonaut training.

In 1974, he commanded his second (and final) space flight Soyuz 14 (Russian: Союз 14) in 1974. Again, his call sign for this flight was Golden eagle (Бе́ркут). This flight was the first to the Salyut 3 space station.

In 1977, he received a post-graduate degree in technical sciences.

In March 1978, he was on duty in the Flight Control Center for Vladimír Remek's flight aboard Soyuz 28.

From 1978 he was the deputy chief of the Gagarin Cosmonauts Training Center responsible for research and testing work. From 1980 to 1989, he was Deputy chief of the Cosmonaut Training Center. In January 1982, he was removed from the list of active cosmonauts, so that he could serve as Deputy Chief for Scientific Testing and Research at the Center.