Background: Routine testing for human immunodeficiency virus (HIV) among pregnant women is done early during antenatal care (ANC) in Tanzania, but retesting for the women found negative is rarely done at term or during delivery. Aim: This study aimed at determining the magnitude and risk factors associated with HIV seroconversion among pregnant women delivering at Bugando Medical Center (BMC). Subjects and Methods: This cross‑sectional study was conducted from January to March 2013 involving 400 pregnant women who tested HIV negative during ANC. These were re‑tested during delivery, and those found positive (and their babies) were given antiretroviral therapy (ART) prophylaxis. All exposed babies were tested by polymerase chain reactions (DNAPCR) at 1 month of age. Sociodemographic and clinical characteristics were collected using a structured questionnaire and patients’ files. Data were analyzed using SPSS version 17.0 software. Results: Of 400 pregnant women (mean age 26.4 [5.73] years) enrolled, HIV seroconversion was found in 5.3% (21/400). Upon multivariate logistic regression analysis, polygamous marriage (P < 0.001) and history of syphilis during ANC visit (P < 0.001) were found to be independent predictors of HIV seroconversion among pregnant women delivering at BMC. One of the 21 babies (4.8%) born of HIV seroconverted women was confirmed to be HIV infected. Conclusion: The high rate of HIV seroconversion found in this study implies that HIV re‑testing should be an enduring exercise. This will allow timely provision of ART prophylaxis to HIV seroconverted women and their exposed babies and thus, prevent mother to child transmission of HIV.