BACKGROUND: HIV prevalence among men who have sex with men (MSM) in Ghana is 18.1% as compared to 1.6% in the general population. Pre-exposure Prophylaxis (PrEP) is recommended by the World Health Organization (WHO) for people who are HIV-negative and at high risk of acquiring HIV. Since PrEP introduction in Ghana in 2020, little is known nationally about the level of awareness, uptake, and willingness to take PrEP among MSM. This study aims to generate estimates on PrEP awareness, uptake and willingness to use among MSM in Ghana. METHODS: We conducted a bio-behavioral survey among MSM aged 18 years and above in all the 10 traditional regions in Ghana from August 2022 to July 2023 using respondent-driven sampling (RDS). RDS-Analyst was used to compute weights based on the participants' network. Data analysis was restricted to MSM who were HIV-negative and sexually active to estimate the prevalence of awareness, willingness, and uptake of PrEP. A multivariable logistic regression model was used to assess the factors influencing these outcomes. RESULTS: Out of the 3,420 total MSM surveyed, 2,627 were HIV negative and were included in the analysis, Out of which 44.5% (95% CI: 42.0-47.0) were aware of PrEP, 90.4% (95% CI: 88.0-92.3) were willing to take PrEP and 17.8% (95% CI: 16.0 - 19.8) had ever taken PrEP. In the regression analyses, PrEP awareness was 5-fold higher among those who completed tertiary education (aOR: 5.56, 95% CI: 2.87-10.78, p <
0.001) and 4-fold among those who interacted with peer educators (aOR: 3.78, 95% CI: 2.52-5.67, p <
0.001). In terms of uptake, the odds were almost 9 times among those who had experienced forced sex (aOR: 8.88, 95% CI: 1.42-55.47, p = 0.02). MSM aged 25-34 were less willing to take PrEP (aOR:0.21, 95% CI 0.07-0.65, p = 0.006) and PrEP use was also less likely among those who consumed high alcohol (aOR:0.42, 95% CI 0.19-0.92, p = 0.03) and never tested for HIV (aOR: 0.44, 95% CI" 0.25-0.88, p = 0.017). CONCLUSION: In Ghana, awareness and willingness to take PrEP to prevent HIV is high, but uptake is low. These results highlight the need for interventions to improve the overall uptake of PrEP among MSM in Ghana, especially among those aged 25-34, high alcohol consumers and those who have never screened for HV. Effective implementation of these findings into the national policies can enhance access and encourage PrEP use, ultimately reducing HIV incidence in Ghana among MSM.