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Many sexually transmitted infections increase risk of mother-to-child transmission (MTCT) of HIV, but the effect of Mycoplasma genitalium is not known. We hypothesized that M. genitalium infection would be common among HIV-infected pregnant women and could be associated with in-utero and intrapartum MTCT.Observational case–cohort study.The current study used specimens from a Kenyan perinatal MTCT cohort (1999–2005) involving HIV-infected women and their infants, who received short-course zidovudine for prevention of MTCT. Vaginal swabs collected at 32 weeks gestation were tested for M. genitalium using a transcription-mediated amplification assay. Infant perinatal HIV infection was determined at birth and 4 weeks of age by DNA PCR. Using a case–cohort design, a random sample was generated with 3 : 1 control : case ratio; prevalence and correlates of M. genitalium were assessed with chi-squared and t tests; predictors of infant outcomes were analyzed using logistic regression.Among 220 HIV-infected pregnant women evaluated, 47 women (21.4%) had M. genitalium. Antenatal M. genitalium infection was associated with higher HIV RNA in plasma (5.0 vs. 4.6 log10 copies/ml in M. genitalium-positive vs. M. genitalium-negative women, P = 0.02) at 32 weeks. Women with M. genitalium were less likely to report prior sexually transmitted infections and genital ulcers (both P = 0.05). There was no association found between exposure to M. genitalium and perinatal MTCT (odds ratio = 0.72, 95% confidence interval 0.35, 1.51, P = 0.39).Vaginal M. genitalium infection was frequently detected among Kenyan HIV-infected pregnant women and was associated with higher plasma HIV levels, but was not associated with perinatal transmission of HIV.