Background HIV disproportionally affects key populations including men who have sex with men (MSM). HIV prevalence among MSM varies from 17% in Brazil and Mexico to 13% in Peru, whereas it is below 0.5% for the general population in each country. Pre-exposure prophylaxis (PrEP) with emtricitabine/tenofovir is being implemented in the context of combination HIV prevention. Reports on willingness to use PrEP among MSM have started to emerge over the last few years. Previously reported factors associated with willingness to use PrEP include awareness, higher sexual risk behavior, and previous sexually transmitted infection. Objective This study aimed to evaluate the factors associated with willingness to use daily oral PrEP among MSM in 3 Latin American, middle-income countries (Brazil, Mexico, and Peru). Methods This Web-based, cross-sectional survey was advertised in 2 gay social network apps (Grindr and Hornet) used by MSM to find sexual partners and on Facebook during 2 months in 2018. Inclusion criteria were being 18 years or older, cisgender men, and HIV-negative by self-report. Eligible individuals answered questions on demographics, behavior, and PrEP (awareness, willingness to use, barriers, and facilitators). Multivariable logistic regression modeling was performed to assess the factors associated with willingness to use daily oral PrEP in each country. Results From a total sample of 43,687 individuals, 44.54% of MSM (19,457/43,687) were eligible and completed the Web-based survey—Brazil: 58.42% (11,367/19,457), Mexico: 30.50% (5934/19,457), and Peru: 11.08% (2156/19,457); median age was 28 years (interquartile range: 24-34), and almost half lived in large urban cities. Most participants were recruited on Grindr (69%, 13,349/19,457). Almost 20% (3862/19,352) had never tested for HIV, and condomless receptive anal sex was reported by 40% (7755/19,326) in the previous 6 months. Whereas 67.51% (13,110/19,376) would be eligible for PrEP, only 9.80% (1858/18,959) of participants had high HIV risk perception. PrEP awareness was reported by 64.92% (12,592/19,396); this was lower in Peru (46.60%, 1002/2156). Overall, willingness to use PrEP was reported by 64.23% (12,498/19,457); it was highest in Mexico (70%, 4158/5934) and lowest in Peru (58%, 1241/2156). In multivariate regression models adjusted for age, schooling, and income in each country, willingness to use PrEP was positively associated with PrEP awareness and PrEP facilitators (eg, free PrEP and HIV testing) and negatively associated with behavioral (eg, concerned by daily pill regimen) and belief barriers (eg, sexual partners may expect condomless sex). Conclusions In this first cross-country, Web-based survey in Latin America, willingness to use PrEP was found to be high and directly related to PrEP awareness. Interventions to increase awareness and PrEP knowledge about safety and efficacy are crucial to increase PrEP dem...