Mei Xiang was put under general anesthesia and inseminated with a combination of fresh semen and frozen semen collected from the zoo's male giant panda, Tian Tian.
The Washington zoo said scientists performed a second and final insemination later on Saturday evening.
"It will be several months before we know if she is pregnant," the National Zoo said in a tweet.
Veterinarians detected a rise in hormone levels on Tuesday, indicating Mei Xiang was ready to breed but said "no competent breeding" between the panda pair had occurred.
"We are hopeful that our breeding efforts will be successful this year, and we're encouraged by all the behaviors and hormonal data we've seen so far," said Dave Wildt, head of the Center for Species Survival at the Smithsonian Conservation Biology Institute.
Scientists will continue to monitor Mei Xiang's hormone levels in the coming months and conduct ultrasounds to determine whether she is pregnant. A pregnancy lasts between 95 and 160 days, they said.
Mei Xiang has given birth to two cubs. One died a week after its birth last year. The other, Tai Shan, was born in 2005 and is now at the China Conservation and Research Center for the Giant Panda in Wolong.
(Reporting by Jane Sutton; editing by Jackie Frank and Sandra Maler)