Despite the well-established protective functions of guilt across childhood, its underlying physiological mechanisms have received little attention. We used latent difference scores (LDS) to model changes in children's (N = 267; 4- and 8-year-olds, 51% girls) skin conductance (SC) and respiratory sinus arrhythmia (RSA) while they imagined themselves committing antisocial acts. We then tested if their later reports of guilt, caregiver-reported aggressive behavior, and age were associated with these physiological changes. For 8-year-olds, changes in RSA leading up to and during transgressions were uniquely associated with the intensity of guilt feelings after transgressions. Eight-year-olds with higher guilt were rated lower in aggression, although children's physiology and aggression were not directly related. We discuss how fluctuations in physiology while transgressing may prepare children to mount adaptive guilt responses afterward and--more broadly--implications for understanding the mechanisms behind guilt and related behavior in early and middle childhood.