Peruvian men who have sex with men (MSM) and transwomen (TW) could benefit from a rectal microbicide (RM) formulated as a rectal douche to prevent HIV infection. However, little is known about rectal douching practices among Peruvian MSM and TW, information necessary to inform RM douche development and future uptake. Using a self-administered interview, we examined the prevalence of and factors associated with rectal douching among a convenience sample of 415 Peruvian MSM and 68 TW. In the previous 6 months, 18 % of participants reported rectal douching using pre-filled commercial kits or plastic bottles or enema bags filled with water, water/soap or saltwater. Multivariate logistic analysis found that “equally insertive and receptive” or “exclusively/mainly receptive” sex roles were associated with douche use. Rectal douching among Peruvian MSM and TW is similar to reports from other studies and supports the potential uptake of a douche-formulated RM in these populations.