University athletes are at high risk for both substance use and mental health problems. This study examined associations between substance use, mental health symptoms, and the resting state functional connectivity (rsFC) of key neural regions involved in self-monitoring and emotional regulation in a sample of female varsity athletes. 31 female university athletes completed measures of substance use, mental health symptoms, and underwent functional MRI scans during the pre-season. Athletes who were substance users had higher symptoms of depression than non-users (p = 0.04; Hedge's g = 0.81). RsFC differences were observed between users and non-users in orbital frontal cortex (OFC) and bilateral hippocampal seeds, and negative associations between depression symptoms and rsFC in the left hippocampus and posterior cingulate cortex were observed in cannabis users. In female athletes, substance use is associated with greater self-reported depression symptoms and altered rsFC in self-monitoring and emotional regulation regions of the brain.