US State Department deputy spokesman Mark Toner made Sunday's call to free those detained on the anniversary of the overnight crackdown on June 3 and 4, 1989.
People's Liberation Army soldiers stormed into central Beijing and fired upon unarmed demonstrators to end six weeks of protests in the square.
More than 1,000 people are believed to have been convicted and jailed in the subsequent clampdown.
"On this the 23rd anniversary of the violent suppression … the United States joins the international community in remembering the tragic loss of innocent lives," said the appeal.
"We encourage the Chinese government to release all those still serving sentences for their participation in the demonstrations; to provide a full public accounting of those killed, detained or missing; and to end the continued harassment of demonstration participants and their families."
Fewer than a dozen people are thought to remain in prison in connection with the protests, including some who are elderly or who are believed to suffer from mental problems.
The appeal also carried a more general call for China to protect "the universal human rights of all its citizens."
A Red Cross report said 727 people were killed in the massacre, while Chinese authorities put the number closer to 200.
Over two decades later, Beijing has refused to apologize for the massacre or consider compensation for those killed. The official view is that the violent crushing of the protests was part of a “counter-revolutionary incident.”
Author: Richard Connor
Editor: Timothy Jones