Background: There is a limited understanding about the impact of spiritual beliefs and activities on HIV seroconversion among black men who have sex with men (BMSM), which we investigate in this study. Setting: United States. Methods: The HIV Prevention Trials Network Study 061 collected demographic and biomedical assessments among BMSM across 6 United States cities for longitudinal analysis. Spiritual beliefs and spiritual activities are constructed composite scales. Bivariate analyses among 894 who provided data at 12-month follow-up compared men who seroconverted to HIV between baseline and 6 months with those who remained uninfected with HIV at 12 months. Cox proportional hazard regression among 944 men tested spiritual beliefs and activities on the longitudinal risk of HIV seroconversion adjusting for age and any sexually transmitted infection (STI). Results: Among this sample, HIV incidence between baseline and 6 months was 1.69%, (95% confidence intervals [CI]: 1.04 to 2.77). Men who seroconverted to HIV were significantly younger than those who remained uninfected at the 12-month follow-up: (mean age 27, SD = 11 vs 37, SD = 12) and a higher proportion reported any STI (46.67% vs 11.39%, P < 0.01). A one-unit increase in spiritual beliefs was associated with lower hazard rate of seroconverting to HIV at follow-up [adjusted hazard ratio (aHR) = 0.37, 95% CI: = (0.16 to 0.87)]. Religious service attendance and spiritual activities were unrelated to seroconverting. Conclusions: Spirituality is important in the lives of BMSM. Biomedical and behavioral HIV prevention interventions should consider assessing spiritual beliefs in HIV care among BMSM.