The purpose of the study was to assess the knowledge and attitude, the level of male involvement and factors associated with male involvement in the prevention of mother-to-child transmission of HIV in Hoima municipality. This study was a descriptive cross section in which quantitative method of data collection was employed in collection of data from respondents. Questionnaires were distributed to participants to assess the knowledge and attitude, the level of male involvement and factors associated with male involvement in prevention of mother-to-child transmission of HIV (PMTCT) in Hoima municipality. Sample size of 200 participants were used, this included the Male partners who hard escorted their pregnant partners to the antenatal clinic aged between 20-50years.The predominant religion were Catholics 59% and seventh day Adventists. Regarding educational levels, majority of respondents had completed secondary level and above (61%) and the predominant ages were between 20-29 years. The study revealed that very few males partner were involved in the PMTCT program especially during HIV counseling and testing (HCT) because of being at old age group above 30years couples, couples not living together, high number of wife’s pregnancies four and above, having no knowledge on methods of MTCT, and husbands failure to discuss HCT with their wives. From the findings, majority of the respondents have ever had about the male involvement in the PMTCT but there was still low male involvement in PMTCT programs at antenatal clinics. There is a need to do an in-depth assessment of women’s experiences when tested HIV-positive in the presence of their partners at the ANC, as well as to develop strategies to improve male involvement. The study again recommends formative research on the use of incentives to promote male involvement in the PMTCT program and the government should train more of the health promoters and the Village Health Teams in order to reach even those that are deep in the village that are not having easy access to the health facility.