BACKGROUND: Tenofovir disoproxil fumarate (TDF) when used as pre-exposure prophylaxis (PrEP) during pregnancy is considered safe overall, however, there is insufficient evidence of its effect on maternal bone. We compared bone mineral density (BMD) and bone mineral content (BMC) at the lumbar spine (LS) and hip of African breastfeeding women exposed and not exposed to TDF-containing PrEP in a randomized control trial (RCT). METHODS: This is a secondary data analysis of an RCT where pregnant women were randomized to initiating PrEP during pregnancy or delayed initiation of PrEP until breastfeeding cessation. BMD and BMC at the LS and hip were measured using dual-energy x-ray absorptiometry (DXA) at 6, 26, 50, and 74 weeks postpartum. In an exploratory analysis, BMD at the hip and LS were evaluated against varying tenofovir levels during pregnancy. RESULTS: Of 300 women in the RCT who had a DXA at 6 weeks postpartum, 102 (66%) women in the Immediate PrEP arm and 105 (72%) in the Delayed PrEP arm had a 74-week DXA scan. Adjusting for breastfeeding duration and body mass index, there were no significant differences in BMD or BMC at the hip and LS between treatment arms. There was no consistent dose-effect of tenofovir diphosphate detected during pregnancy on BMD at the hip ( P = 0.231) or the LS ( P = 0.277). CONCLUSIONS: After adjusting for breastfeeding and body mass index, tenofovir disoproxil fumarate when given as oral PrEP during pregnancy had no deleterious effect on BMD and BMC at the hip and LS of African breastfeeding women.