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Background: Mother-to-child transmission (MTCT) of HIV continues to be a major public health problem and accounts for a considerable share of new HIV infections among young children. Prevention of mother-to-child transmission (PMTCT) has been recognised as vital to reducing infant and child mortality associated with HIV infection. This study assessed the utilization and implementation challenges of PMTCT of HIV programme in a secondary care hospital in Ghana. Methods: We conducted an institution based cross-sectional survey in April 2015 using exit interviews with 135 pregnant women attending antenatal care (ANC) clinic of Bolgatanga Regional hospital of Upper East Region, Ghana. Pre-tested questionnaires were administered to collect data on socio-demographic characteristics, knowledge about MTCT of HIV, and HIV counselling and testing practice. Six in-depth interviews with service providers were conducted to assess PMTCT programme implementation challenges. Antenatal and PMTCT records were reviewed to evaluate PMTCT utilization. Stata version 11 was used to analyse quantitative data. Chi-square test was performed to test for differences between proportions for the cross tabulated variables. Level of significance was set at p< 0.05. Results: Fifty-six (41.5%) of participants made up to four or more ANC visits. The majority (62; 45.9%) were in the third trimester of gestation, 75 (56%) were primigravida and 12 (9%) ever had four or more pregnancies. About 96%(129) visited the health facility for ANC check-up. Only 3 (2.2%) visited the health facility for HIV counselling and testing. The majority (112; 83%) were aware of …