Introduction: Mother–to-child transmission of HIV is a major public health problem in Cameroon. The aim of this study was to assess knowledge of pregnant women on HIV transmission and prevention, particularly the four pillars of mother-to-child transmission. Materials and Methods: This was a descriptive cross-sectional study from October 2008 to January 2009 at the Yaoundé Gynaecology-Obstetrics and Pediatric Hospital. All women presenting at the Ante-Natal Care (ANC) Clinic for the first time were included in the study after obtaining a verbal informed consent. Results: The ages of the 260 women included in the study ranged from 15 to 42 years. Almost 99% (257/260) of the women interviewed had heard about HIV. Respectively, 80.5% (209/260), 89.3% (232/260) and 81.2% (211/260) of the women cited pregnancy, delivery, and breastfeeding as risk periods for HIV transmission from mother to child. Use of the male condom, the female condom, abstinence, and faithfulness to a single partner were considered as effective methods of HIV prevention by 73% (190/260), 76% (198/260), 88% (229/260) and 46% (120/260) of respondents respectively. About 79% (64/81) of participants with higher education considered HIV infection to be contagious as opposed to 45.5% (5/11) of women with no formal education (P = 0.008). Conclusion: Our data suggest that women have some good knowledge on the Prevention of Mother-To-Child Transmission (PMTCT) of HIV. Nevertheless, improving the formal educational level of these women may contribute to a further reduction of HIV transmission