Abstract In a sample of men who have sex with men (MSM) (N = 3436) in Spain who bear intrinsic HIV risk, we investigated how internalised homonegativity (IH) is associated with the number of non-steady male partners with condomless intercourse (as a proxy of sexual risk behaviour). Using structural equation modelling (SEM), we examined the relationship between IH and sexual risk behaviour, and mediating effects of HIV/PrEP knowledge and substance use during sex on this relationship. We found no direct association between IH and sexual risk behaviour, nor did IH influence substance use during sex. In line with our hypothesis, association between IH and sexual risk behaviour was significant when mediated by HIV/PrEP knowledge. We found that as IH increased, sexual risk behaviour decreased, because higher IH was associated with lower HIV/PrEP knowledge while higher HIV/PrEP knowledge was associated with increased non-condom use with non-steady partners. Substance use during sex was significantly associated with sexual risk behaviour. Our results emphasize the continuing importance of prevention strategies focused on behavioural changes and community level interventions, especially targeting substance use.