BACKGROUND: To effectively reduce vertical HIV transmission requires a reduction of HIV prevalence and incidence among pregnant women and a full understanding of its epidemiology. The study aimed to determine the prevalence of HIV among women attending antenatal care and further determine spousal support during antenatal care attendance in rural areas in Eastern Cape province, South Africa. METHODS: A Cross-sectional study of women attending antenatal care in four Primary Care facilities was conducted using an interviewer-administered questionnaire which collected information on socio-demographic characteristics and medical history. Binomial logistic regression analyses were used to determine factors associated with HIV and to estimate the prevalence ratio (PR). The 95% confidence interval (95%CI) is used for precision of estimates; p≤0.05 for statistical significance. RESULTS: A total of 343 participants were included in the final analysis. The antenatal HIV prevalence was 38.2% (95%CI: 33.2-43.9). For 75% of the women, the HIV diagnosis was made 141 days before the date of the interview (median=77 days, interquartile range=42-141 days). Participants between the age of 30 to 39 years were 50% more likely to be HIV positive compared to those who were between the age of 20 to 29, these differences were statistically significant (PR=1.5; p-value=0.001). Furthermore, self-employed women were 30% less likely to be HIV positive when compared to unemployed participants, this was also statistically significant (PR=0.7; p-value< 0.0001). CONCLUSION: Despite a 100% antenatal HIV testing rate, the antenatal HIV prevalence remains high in this population, coupled with no spousal attendance in antenatal care. It is important to move beyond awareness about the HIV status to actionable strategies of reducing the HIV incident cases. It is therefore important to remain vigilant and monitor mother-to-child transmission that could be associated with this increased prevalence.