Little is known about men's potential motivations and barriers associated with specific sexually transmitted infections (STI) testing methods. In this study, we examined experiences of self-sampling for ano-rectal STI among men who have sex with men (MSM) in a midwestern community in the U.S. A total of 75 MSM were recruited from community venues throughout Indianapolis, Indiana. Participants completed semi-structured interviews, were asked to obtain ano-rectal self-sample in a private restroom, and were asked open-ended questions about their experiences with ano-rectal self-sampling for STI. Participants included 35 White, 27 Black, and 13 Latino MSM who ranged in age from 18 to 57 years. Regardless of sexual practices, most participants who obtained an ano-rectal self-sample (68/75) reported that the sampling procedure was relatively painless and physically easy. However, regardless of previous receptive anal sex, participants also expressed concerns about the nature of the test (i.e., inserting something into their rectum), which required increased levels of privacy and cleanliness compared to collection of urine samples. Self-sampling proved to be a feasible and acceptable method of collecting ano-rectal STI specimens among MSM. Increased testing for ano-rectal STI among MSM may require addressing the location of sampling and testing sites, existing negative perceptions of ano-rectal self-sampling, and the measures in place to promote privacy and cleanliness.