Prostate cancer accounts for 20.3% of all cancers in men in sub-Saharan Africa. Early screening among at-risk groups is challenging in Uganda, with limited data on prostate cancer screening uptake in most districts, including newly established ones. The purpose of this study was to determine factors influencing the uptake of prostate cancer screening among men aged≥40 in Kazo Town Council, Kazo District, a newly created district. We used a descriptive cross-sectional study design that employed both quantitative and qualitative data collection methods. Participants were recruited through simple random sampling between November 2020 and January 2021. Structured questionnaires were used for quantitative data (n=300). Statistical analyses to determine associations were carried out using inferential and chi-square tests followed by logistic regression. In-depth interviews were conducted with 10 key informants and analyzed thematically to explore a range of perceptions related to prostate cancer screening. Only 10 (3.33%; 95% CI: 0.018-0.60) respondents had ever screened for prostate cancer. Lack of privacy (p<0.033), access to prostate cancer information (p<0.014), and distance to health facilities (p<0.001) were significantly associated with the uptake of prostate cancer screening. Marital status (OR=7.93; 95% CI: 1.85-33.99; p=0.005), positive health worker attitudes (OR=0.002; 95% CI: 0.000-0.023, p<0.001), and perceived affordability (OR=0.001; 95% CI: 0.000-0.011, p<0.001) were independently associated with uptake of prostate cancer screening. Key barriers included lack of information, access to screening centres, and fear of screening. The level of uptake of prostate cancer screening was considerably low among men aged 40 and above in the Kazo District. Targeted community interventions to improve access to prostate cancer information, screening, sensitization, and addressing perceived and actual barriers are needed in newly created districts to bolster the uptake of prostate cancer screening. This has implications for prioritizing research evaluating district resource allocation to support optimized and integrated evidence-based service delivery in primary healthcare centres, especially for specialized services in newly created districts.