Edinburgh Zoo panda keepers were hopeful that Tian Tian would mate with male Yang Guang this year, but moved on to artificial insemination after her hormone levels started to fall quickly.
The procedure was carried out on Sunday using samples from Yang Guang.
Experts said they will not know for certain whether Tian Tian is pregnant until August or September.
Their hopes of a pregnancy follow last year's disappointment when the pair did not mate. Although Tian Tian was artificially inseminated, she lost her foetus at late term.
Iain Valentine, director of giant pandas for the Royal Zoological Society of Scotland (RZSS), said: "From the start, when the pandas started to show breeding behaviour early this spring, both were showing very positive signs.
"We were hopeful natural mating would occur this year, but in the end Tian Tian's hormones started to fall quickly, which meant her breeding window could be much shorter. Although our Chinese expert Dr Wang Chengdong, from the China Conservation and Research Centre for Giant Pandas, was confident the pair would mate naturally, after the first unsuccessful introduction attempt, time restrictions meant we needed to move quickly to artificial insemination."
Experts at Edinburgh Zoo monitored Tian Tian's hormones to establish when she would enter her brief fertile period.
They also watched her behaviour closely to gauge when her 36-hour breeding window would begin.
The zoo said both pandas recovered well from the artificial insemination and were up and about soon afterwards, with Yang Guang enjoying honey and bamboo 15 minutes later.
Mr Valentine said: "The panda enclosure will remain closed to the public until Wednesday, but both pandas have been out and about, are eating well and are in good health. As giant pandas experience pseudo pregnancies and delayed implantation, it is very likely we will not 100% know if Tian Tian is pregnant until she gives birth. This is usually August to September but can continue much later, as we saw last year."
The panda pair, now 10, arrived on loan from China in December 2011 and will remain at Edinburgh Zoo for a decade. The zoo said the panda breeding programme can play an important role in conservation.
Chris West, chief executive of the RZSS, said: "If we can successfully assist Tian Tian and Yang Guang to breed, we will be adding to the total number of pandas in zoos around the world and in breeding centres in China. The more there are, the greater and more diverse the gene pool is from which pandas can be selected for re-introduction."