Background: Brazil has a lot of people living with HIV (PLWH) that are still unaware of their own serostatus and has recently integrated HIV self-test (HIVST) into its public health system and offered to key populations such as men who have sex with men (MSM). This study estimates HIVST acceptability among Brazilian MSM and its associated factors. Methods: Respondent-driven sampling (RDS) was used in behavioral and biological surveillance to recruit 4,176 MSM in 12 Brazilian cities in 2016. Descriptive, bivariate and multivariate analyses were conducted. Overall proportions were weighted according to Gile’s estimator using RDS Analyst Software and 95% confidence intervals were calculated. The analyses of HIVST acceptability were stratified by prior HIV testing (never or at least once in a lifetime). Results: We included 3,605 MSM who did not previously know they were PLWH. The HIVST acceptability was 49.1%, lower among those had done HIV test during lifetime compared to those who have never done it (42.7% vs 50.1%), especially among who are in a less vulnerable social context. In the subgroups of MSM who had never had an HIV test, those with complete secondary or incomplete higher school, who reported discrimination, who with a medical appointment in the last 12 months, with high levels of knowledge of HIV/AIDS and who took part in LGBT nongovernmental organization (LGBT-NGO) had higher HIVST acceptability. Among MSM who had taken an HIV test, those who took part in LGBT NGOs had greater HIVST acceptability. We observed a dose-response effect of schooling and knowledge of HIV/AIDS on HIVST acceptability: an increase in both levels was associated with greater acceptability. Conclusions: The findings highlight that it is necessary to improve information about HIVST among MSM more vulnerable in Brazil. Given the acceptability of self-testing and its current adoption 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.