Background: Brazil has a substantial population living with HIV (PLWH) who are still unaware of their serostatus and has recently added HIV self-testing (HIVST) into its public health system for key populations such as men who have sex with men (MSM). This study estimates HIVST acceptability among Brazilian MSM and its associated factors among those who had never been tested for HIV or had a previous HIV negative result.Methods: Respondent-driven sampling (RDS) was used for behavioral and biological surveillance to recruit 4,176 MSM in 12 Brazilian cities in 2016. We excluded all MSM who were aware of their positive HIV serostatus before the study. Descriptive, bivariate and multivariate analyses were conducted. Overall proportions were weighted with Gile’s estimator in RDS Analyst software and 95% confidence intervals were calculated. The analyses of HIVST acceptability were stratified by prior HIV testing (never or one or more times).Results: For this analysis, 3,605 MSM unaware of their HIV serostatus before the study were included. The acceptability of HIVST was 49.1%, lower among those had never tested for HIV (42.7%) compared to those who had a previous HIV negative test (50.1%). In the subgroup of MSM who had never had an HIV test, those who reported discrimination and who had a medical appointment in the last 12 months reported higher HIVST acceptability. Among MSM who had a previous HIV negative test, only those reporting condomless receptive anal sex had greater HIVST acceptability. In addition, we observed that high levels of knowledge of HIV/AIDS, taking part in lesbian, gay, bisexual, and transgender nongovernmental organizations (LGBT-NGO), and complete secondary or incomplete higher school were associated with greater acceptability in both groups. Conclusions: The acceptability of HIVST was low among MSM, especially among those had never tested for HIV. Given the acceptability of HIVST and its current promotion in Brazil, we can also point to the need for policies that build on this and enhance timely access to health services and prevention information.