Background: Indonesia is the only country in the Asia Pacific region where the incidence of HIV is still on the rise, and its prevalence is extremely high among the key populations such as men who have sex with men, transgender women, and people who inject/use drugs. Mobile health (mHealth) apps provide an innovative platform for delivering tailored HIV prevention and care among these populations more efficiently than possible through the direct face-to-face approach., Objective: The aim of this study was to assess the role of a peer-customized mobile app based on the principle of self-learning for improving HIV prevention knowledge and access to health services among men who have sex with men, transgender women (known as Waria in Indonesia), and people who use drugs in Indonesia., Methods: A prospective intervention cohort study was conducted among the key populations in five provinces of Indonesia (Jakarata, West Java, East Java, Special Region of Yogyakarta, and Bali). The data were evaluated using a pre-post assessment survey conducted on a sample of 200 unique users, including 50 men who have sex with men and transgender women each, and 100 people who use drugs, with a follow-up response rate of 98% and 70%, respectively. An mHealth app named RUMAH SELA was developed and implemented among the key populations., Results: From baseline to the endpoint of the study, there was a significant increase in comprehensive HIV-related knowledge from 20% (10/49) to 60% (29/49), 22% (11/49) to 57% (28/49), and 49% (34/70) to 74% (52/70) among men who have sex with men (P=.004), transgender women (P<.001), and people who use drugs (P<.001), respectively. There was also a reduction in sexual activities without condom use from 22% (11/49) to 19% (9/49), 18% (9/49) to 12% (6/49), and 21% (15/70) to 10% (7/70) among men who have sex with men (P=.45), transgender women (P=.25), and people who use drugs (P<.001), respectively. There was an uptake of HIV testing by 31% (15/49) for men who have sex with men, 49% (24/49) for transgender women, and 26% (18/70) for people who use drugs after using the app. There was a reduction in injecting drugs with a used needle in drug users from 45/70 (78%) to 15/70 (26%). Measures of self-esteem increased among men who have sex with men (mean 26.4 vs mean 27.1), transgender women (mean 26.5 vs mean 27.8; P=.02), and people who use drugs (mean 24.0 vs mean 25.0). In addition, 27% (7/24) of men who have sex with men, 25% (4/15) of transgender women, and 11% (2/18) of drug users made an appointment for an HIV test through the app. The app was quite highly accepted by the key populations as nearly a quarter felt that they became more confident in discussing issues about sexuality, more than 80% found that the app provided sufficient knowledge about HIV, and more than half of the participants found the app to be user friendly., Conclusions: This one-of-a-kind mHealth intervention with an mHealth app as a self-learning tool is effective in increasing HIV-related knowledge and behavior, and access to services with strong acceptability by the community. There is a need to scale up such interventions for efficacy testing in a larger population to provide evidence for national-level mHealth programs addressing HIV., (©Priyanka Rani Garg, Leena Uppal, Sunil Mehra, Devika Mehra. Originally published in JMIR mHealth and uHealth (http://mhealth.jmir.org), 08.09.2020.)