Sister Maria Gomez Valbuena, 80, is accused of stealing a mother's newborn daughter at a Madrid hospital in 1982.
Appearing before a judge on Thursday, Sister Maria refused to testify.
Thousands of babies are thought to have been taken from parents in hospitals and given to other families during the Franco dictatorship and later.
The alleged thefts are thought to have started in the 1936-39 Civil War. Some cases are believed to happened as late as the 1990s.
The practice is believed to have been motivated by a desire to remove children from "undesirable" left-wing parents and give them to "approved" right-wing families.
The Spanish authorities have investigated hundreds of complaints related to the events. Many parents say they were told by hospital staff that their babies had died during or after childbirth.
Many cases have been dismissed for lack of evidence.
Clad in the habit of her Sisters of Charity order, Sister Maria was jeered as she was escorted out of court in Madrid.
The mother in Sister Maria's case, Maria Luisa Torres, says the nun took away her baby girl soon after she gave birth in the Santa Cristina hospital, where Sister Maria worked.
Ms Torres has told the court that when she asked Sister Maria where the missing baby had gone, the nun threatened to take away another daughter and have Ms Torres jailed for adultery.
Ms Torres was reunited with her daughter, Pilar, last year.
The formal charges Sister Maria faces in relation to the case are false imprisonment and forgery.
A Spanish nun has become the first person to appear in court in connection with the alleged theft of newborn babies, mostly in the Franco era.