Alcohol and cannabis use are highly prevalent among adolescents and associated with negative consequences. Understanding motivations behind substance use in youth is important for informing prevention and intervention efforts. The present study aims to examine negative reinforcement principles of substance use among adolescent cannabis and alcohol users by pairing a cue reactivity paradigm with an aversive interoceptive stimulus. Adolescents (ages 15–17), classified as controls (CTL; n = 18), cannabis and/or alcohol experimenters (CAN+ALC-EXP; n = 16), or individuals meeting clinical criteria for cannabis and/or alcohol use disorder (CAN+ALC-SUD; n = 13) underwent functional magnetic resonance imaging during which they experienced an aversive interoceptive probe delivered via breathing load while simultaneously performing a cue reactivity paradigm. Participants also provided self-report ratings of how their substance use is positively or negatively reinforced. While experiencing the breathing load, CAN+ALC-SUD exhibited greater (p < 0.05) deactivation in the right amygdala, the left inferior frontal gyrus, and the left parahippocampal gyrus than CAN+ALC-EXP and CTL, who did not differ. Across all substance users, activation during the breathing load within the left parahippocampal gyrus negatively correlated with cannabis and alcohol lifetime use episodes and the left inferior frontal gyrus activity negatively correlated with lifetime alcohol use episodes. CAN+ALC-SUD reported experiencing more positive and negative reinforcement of using their substance of choice than CAN+ALC-EXP; both user groups reported higher levels of positive than negative reinforcement. Adolescents with a cannabis/alcohol use disorder demonstrate an altered response to interoceptive perturbations. However, adolescent cannabis/alcohol use does not appear to be driven by negative reinforcement, as viewing substance images did not dampen this response. Based on self-report data, the experience of positive reinforcement may be stronger for adolescents. Future studies should examine whether positive reinforcement contributes to adolescent substance use.