We conducted 3 studies to test the idea that guilt is a key affective component of Conscientiousness and that it can account for the relation between Conscientiousness and negative affect. Study 1 used meta-analysis to show that Conscientiousness was associated with specific emotions and overall negative affect but was most strongly associated with guilt. Conscientiousness was negatively related to guilt experience but positively related to guilt proneness. Also, guilt experience mediated the relation between Conscientiousness and negative affect. Study 2 (N = 142) examined the relation between facets of Conscientiousness and guilt. We replicated results from Study 1 and showed that the relation between Conscientiousness and guilt was not due to overlap with Extraversion and Neuroticism. Study 3 (n = 176) examined the interplay between Conscientiousness and guilt on grades in a short-term longitudinal study. These studies showed that Conscientiousness is primarily related to guilt and highlighted the importance of examining the emotional substrate of Conscientiousness.