Indicators of risk for developing Autism Spectrum Disorder (ASD) are difficult to detect within the first year of life. In this study, infants who were originally followed prospectively to examine general developmental risks due to substance exposure interacted with their mother and an unfamiliar experimenter for 2-min episodes at 4 months of age. Electrocardiogram was collected to measure respiratory sinus arrhythmia (RSA) and the session was video recorded for behavioral coding. Two groups of infants were compared: infants with a diagnosed ASD outcome (N = 8) and matched controls (N = 186). Infants were compared on mean RSA and infant behavioral codes for each 2-min episode. No significant group differences were revealed on RSA or behavior during interactions between the infants and mothers. However, in response to play with a stranger, infants with an ASD outcome had lower RSA (M = 2.49, 95% CI [2.30, 2.98]) than controls (M = 3.12, 95% CI [3.06, 3.18]). During the interaction with a stranger, lower RSA response was associated with more protesting behaviors (P < 0.01), whereas higher RSA response was associated with more social monitoring (P = 0.001). Lower RSA suggests that 4-month-old infants later diagnosed with ASD exhibited poorer autonomic regulation during interaction with an unfamiliar adult than did controls. Physiological regulation during interactions with a new social partner may be disrupted early in infancy in children with ASD, as indicated here by lower RSA, and therefore be a potential target for developing early risk screening tools for ASD. Autism Res 2018. © 2018 International Society for Autism Research, Wiley Periodicals, Inc.