BACKGROUND : Recently, we reported that the microRNAs (miRNAs) miR-20a-5p and—to a lesser extent—miR-222-3p hold potential as biomarkers for gestational diabetes mellitus (GDM) in human immunodeficiency virus (HIV)-negative South African women. METHODS : In this preliminary study, we measured the expression of these miRNAs in HIV-positive women (GDM 15, non-GDM 52; median 26.0 weeks; range 16–30). RESULTS : Although the same trend of decreased expression of miR-20a-5p (1.5-fold decrease) and miR-222-3p (1.4-fold decrease) was observed in sera of women with and without GDM, these differences were not statistically significant. Stratification according to antiretroviral treatment (ART) confirmed decreased expression of miR-20a-5p and miR-222-3p in ART-naïve and ART-treated women with GDM, although again this was not statistically significant. CONCLUSION : Our results demonstrate that HIV infection modifies the expression of miR-20a-5p and miR-222-3p in women with GDM. Importantly, this study highlights the complexities of miRNA profiling and the need for GDM biomarker discovery in both HIV-infected and uninfected individuals, particularly in South Africa, where approximately 30% of pregnancies are complicated by HIV. Further studies to elucidate the mechanisms that underlie these miRNA differences are needed.