The feeling of guilt is a complex mental state underlying several human behaviors in both private and social life. From a psychological and evolutionary viewpoint, guilt is an emotional and cognitive function, characterized by prosocial sentiments, entailing speciﬁc moral believes, which can be predominantly driven by inner values (deontological guilt) or by more interpersonal situations (altruistic guilt). The aim of this study was to investigate whether there is a distinct neurobiological substrate for these two expressions of guilt in healthy individuals. We ﬁrst run two behavioral studies, recruiting a sample of 72 healthy volunteers, to validate a set of stimuli selectively evoking deontological and altruistic guilt, or basic control emotions (i.e., anger and sadness). Similar stimuli were reproduced in a event-related functional magnetic resonance imaging (fMRI) paradigm, to investigate the neural correlates of the same emotions, in a new sample of 22 healthy volunteers. We show that guilty emotions, compared to anger and sadness, activate speciﬁc brain areas (i.e., cingulate gyrus and medial frontal cortex) and that different neuronal networks are involved in each speciﬁc kind of guilt, with the insula selectively responding to deontological guilt stimuli.