We examined whether and how regret contributes to acquiring autonomous motivation with the aim of gaining a deeper understanding of the intrapersonal process of the internalization of academic motivation. We conducted a longitudinal survey to examine the longitudinal relationship between motivation and regret in academic situations. Results of a path analysis showed that regret about neglecting study, experienced immediately after an end-of-term examination, mediated the conversion of controlled (especially, introjected) motivation into autonomous (i.e., intrinsic and identified) motivation. In contrast, participants' regret about not having enjoyed themselves in the long term negatively predicted autonomous motivation in a subsequent examination. These results indicated that participants' regret about neglecting their studies contributed to internalization, but regret about not having enjoyed themselves interfered with this. We discussed new insights for both educational practices and psychological theories.