In this study, we investigated, for the first time, facial expressions during the retrieval of Self-defining memories (i.e., those vivid and emotionally intense memories of enduring concerns or unresolved conflicts). Participants self-rated the emotional valence of their Self-defining memories and autobiographical retrieval was analyzed with a facial analysis software. This software (Facereader) synthesizes the facial expression information (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). We found that participants showed more emotional than neutral facial expressions during the retrieval of Self-defining memories. We 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 emotional subjective experience of Self-defining memories. These findings provide valuable physiological information about the emotional experience of the past.