Whether internalizing symptoms increase or remain at similar levels throughout childhood is currently not well understood. Moreover, the association between vagal regulation of cardiac activity and internalizing symptoms across childhood needs to be clarified. We used a multilevel conceptual framework to examine how children's vagal regulation of cardiac activity and mothers' internalizing symptoms were jointly associated with children's developmental trajectories of internalizing symptoms from ages 4 to 10 years old. Data came from 384 children who participated in an ongoing longitudinal study. Children and their mothers came to the research laboratory at ages 4, 5, 7, and 10. Mothers reported their children's and their own internalizing symptoms. Children's vagal regulation of cardiac activity was assessed during quiet baseline tasks and during challenge tasks. Multilevel models revealed that child internalizing symptoms increased from ages 4 to 10 years old, but only in females, and especially between ages 7 and 10. More vagal withdrawal in response to challenge was associated with more internalizing symptoms, particularly with more somatic symptoms. Associations between children's physiological regulation and internalizing symptoms differed by children's age, sex, and presence of maternal internalizing symptoms. Understanding associations between vagal regulation of cardiac activity and internalizing symptoms during childhood calls for fine-grained developmental analyses that take into account the heterogeneity of internalizing symptoms, as well as developmental phase, context, and gender.