Maternal substance use may interfere with optimal parenting, lowering maternal responsiveness during interactions with their children. Previous work has identified maternal autonomic nervous system (ANS) reactivity to parenting-relevant stressors as a promising indicator of real-world parenting behaviors. However, less is known about the extent to which individual differences in emotion dysregulation and reward processing, two mechanisms of substance use, relate to maternal ANS reactivity in substance-using populations. The current study examined associations among emotion dysregulation, reward responsiveness, and ANS reactivity to an infant cry task among 77 low-income and substance-using women who were either pregnant (n = 63) or postpartum (n = 14). Two indicators of ANS functioning were collected during a 9 min computerized infant cry task (Crybaby task): respiratory sinus arrhythmia (RSA) and pre-ejection period. Mothers also completed self-reported measures of emotion dysregulation and reward responsiveness. Analyses revealed that trait emotion regulation was associated with RSA reactivity to the Crybaby task, such that greater emotion dysregulation was associated with greater RSA reduction during the infant cry task than lower emotion dysregulation. Reward responsiveness was not significantly associated with either indicator of ANS reactivity to the task. Findings revealed distinct patterns of associations linking emotion dysregulation with ANS reactivity during a parenting-related computerized task, suggesting that emotion regulation may be a key intervention target for substance-using mothers.