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Mycoplasma genitalium is an emerging sexually transmitted infection (STI) and has been associated with reproductive tract infections and HIV in cross-sectional studies. In this longitudinal study, we assess whether M. genitalium is associated with risk of acquiring HIV-1 infection.Nested case–control study within a large prospective study in Zimbabwe and UgandaA total of 190 women who seroconverted to HIV-1 during follow-up (cases) were matched with up to two HIV-negative controls. Mycoplasma genitalium testing was performed by PCR-ELISA, using archived cervical samples from the HIV-1 detection visit and the last HIV-negative visit for cases, and equivalent visits in follow-up time for controls. Risk factors for HIV-1 acquisition were analyzed using conditional logistic regression, with M. genitalium as the primary exposure.Mycoplasma genitalium was a common infection in these populations (14.8 and 6.5% prevalence among cases and controls, respectively, at the visit prior to HIV-1 detection), and more prevalent than other nonviral STIs. We found a greater than two-fold independent increased risk of HIV-1 acquisition among women infected with M. genitalium at the visit prior to HIV-1 acquisition [adjusted odds ratio (AOR) = 2.42; 95% confidence interval (CI) 1.01–5.80), and at time of HIV-1 acquisition (AOR = 2.18; 95% CI 0.98–4.85). An estimated 8.7% (95% CI 0.1–12.2%) of incident HIV-1 infections were attributable to M. genitalium.This is the first longitudinal study to assess the relationship between M. genitalium and HIV-1 acquisition. If findings from this research are confirmed, M. genitalium screening and treatment among women at high risk for HIV-1 infection may be warranted as part of an HIV-1 prevention strategy.