Substance use and depression are prevalent in sexual and gender minorities (SGM), but evidence about their impacts on HIV pre-exposure prophylaxis (PrEP) use is mixed. We assessed these impacts in a US-based cohort of 3,330 young SGM who tested negative for HIV and completed baseline and semiannual assessments on substance use (cocaine, methamphetamine, or heroin), depression, and PrEP use and adherence. We estimated prevalence differences (PDs) to compare baseline and 12-month PrEP use and adherence between participants with and without substance use and depression, separately and jointly. At baseline, 8% of participants reported substance use and depression, 7% reported substance use only, 38% reported depression only, and 47% reported neither. PrEP use was reported by 22%, with 78% reporting prior-day adherence. Compared to participants without depression or substance use, participants with substance use alone were more likely to report PrEP use at baseline (PD = 18.9; 95% CI: 7.6, 30.2) and 12 months (PD = 15.9, 95% CI: 5.1, 26.7), without adherence differences. Depression alone was associated with lower baseline PrEP use (PD=-3.6, 95% CI: -6.7, -0.4) and adherence (PD=-10.0, 95% CI: -18.4, -1.6), but associations attenuated over 12 months. Those with both substance use and depression (vs. neither) reported higher baseline PrEP use (PD = 6.5, 95% CI: -0.6, 13.5) and lower adherence (PD=-12.9, 95% CI: -26.2, 0.5), with associations weakening over 12 months. Substance use and depression had opposing relationships with PrEP use and adherence. In addition to its mental health benefits, depression treatment may improve PrEP uptake and adherence in young SGM.