Early substance use is associated with long-term negative health outcomes. Emotion regulation (ER) plays an important role in reducing risk, but detecting those vulnerable because of ER deficits is challenging. Respiratory sinus arrhythmia (RSA), a biomarker of ER, may be useful for early identification of substance use risk. To examine this, we enrolled 23 adolescents (Mage = 14.0; 56% minority) with and without a history of substance use and collected RSA during a neutral baseline, virtual reality challenge scene, and neutral recovery. ANOVAs indicated that adolescents who reported having used a substance were not different from non-using peers on baseline or challenge RSA but demonstrated lower RSA during recovery. This suggests that adolescents with a history of substance use exhibit slower return to baseline RSA after experiencing a challenging situation compared to non-using peers. RSA, an index of ER, may be useful in identifying adolescents at risk for early substance use.