The aim of the study is to assess associations between substance use and menopausal symptoms among US people living with and without HIV in a longitudinal cohort. We analyzed self-reported menopausal symptoms and substance use from biannual Women's Interagency HIV Study (WIHS) visits from 2008-2020. Substance use since the last visit or lifetime cumulative use included tobacco, alcohol, marijuana, crack/cocaine, and opioids. Logistic regression quantified associations between each substance use and menopausal symptom frequency (vasomotor, mood, and musculoskeletal), adjusting for other substance use, HIV status, demographics, comorbidities, and trauma. A total of 1,949 participants contributed early perimenopausal, late perimenopausal, or postmenopausal study visits. Across reproductive-aging stages, based on menstrual history, and among participants with and without HIV, participants reported frequent vasomotor (range 22-43%), mood (18-28%), and musculoskeletal (25-34%) symptoms. Many reported ever using tobacco (72%), heavy alcohol (75%), marijuana (73%), crack (50%), and opioids (31%). Current heavy alcohol use (odds ratio [OR]: 1.22; 95% confidence interval [CI]: 1.10-1.37), cumulative marijuana use (OR: 1.15, 95% CI: 1.01-1.32), and cumulative tobacco use (OR: 1.06, 95% CI: 1.01-1.12) were associated with a higher frequency of vasomotor symptoms; current heavy alcohol use (OR: 1.20, 95% CI: 1.04-1.39) and current opioid use (OR: 1.13; 95% CI: 1.01-1.25) were associated with mood symptoms; and current opioid use (OR: 1.11, 95% CI: 1.00-1.23) was associated with musculoskeletal symptoms. All other associations were found to be null. Current and prior substance use may independently affect symptoms experienced during the menopausal transition and may indicate potential to benefit from additional intervention and referral to menopause specialty care.