This study, for the first time investigates facial expressions during the retrieval of self-defining memories (i.e., vivid and emotionally intense memories of enduring concerns or unresolved conflicts). Participants self-rated the emotional valence of their self-defining memories, while autobiographical retrieval was analyzed with facial analysis software. This software (Facereader) synthesizes the information in facial expression (i.e., cheek, lips, muscles, eyebrow muscles) to describe and categorize facial expressions (i.e., neutral, happy, sad, surprised, angry, scared, and disgusted facial expressions). It was found that participants showed more emotional than neutral facial expressions during the retrieval of self-defining memories. It was also found that participants showed more positive than negative facial expressions during the retrieval of self-defining memories. Interestingly, participants attributed positive valence to the retrieved memories. These findings are the first to demonstrate the consistency between facial expressions and the subjective emotional experience of self-defining memories. These findings provide valuable physiological information about the emotional experience of the past.