Background: Human Immunodeficiency Virus (HIV) remains a significant public health concern in sub-Saharan Africa, particularly among pregnant women due to the risk of adverse pregnancy outcomes and mother-to-child transmission (MTCT). Although Ghana has implemented prevention strategies such as Prevention of Mother-to-Child Transmission (PMTCT) programs and antiretroviral therapy (ART), localized data are needed to guide districtlevel interventions. This study assessed the prevalence and determinants of HIV infection among pregnant women attending antenatal care (ANC) in the Akuapem North Municipality, Ghana. Results: A total of 912 ANC records were reviewed. The overall HIV prevalence among pregnant women was 1.97% (95% CI: 1.25–3.11). HIV infection was significantly associated with age group (p = 0.001) and place of residence (p = 0.004), with higher prevalence observed among women aged 20–24 years and those residing in rural communities. HIV prevalence showed a declining trend over the six-year period, decreasing from 3.42% in 2019 to 1.06% in 2024, reflecting improvements in HIV prevention and treatment programs. Gestational age, parity, and gravidity were not significantly associated with HIV infection. Conclusion: HIV prevalence among pregnant women in the Akuapem North Municipality is relatively low and has declined over time, suggesting progress in PMTCT and HIV control efforts. However, young women and those in rural areas remain disproportionately affected. Strengthening targeted health education, expanding rural HIV testing services, and improving access to antenatal HIV care are essential to sustain progress toward the elimination of motherto-child transmission of HIV in Ghana.