Aims It is reported that elevated homocysteine (Hcy) level represents an independent risk factor for gestational diabetes mellitus (GDM). However, the relationship between Hcy level and GDM remains controversial. Our study aimed to systematically review available literature linking Hcy to GDM for a comprehensive understanding of the relationship between circulating Hcy level and GDM in humans.Methods PubMed, The Cochrane Library, and Web of Science were searched for studies published up to January 2021. Manual searches of references of the relevant studies were also conducted. Standard mean difference (SMD) with 95% confidence interval (95%CI) were calculated to evaluate the relationship between Hcy level and GDM using the Review Manager 5.3 and Stata 12.0.Results Of 106 references reviewed, 12 studies with a total of 712 GDM patients contributed to the present meta-analysis. Hcy level was significantly elevated in women with GDM compared with those without GDM (SMD = 0.55; 95% CI: 0.25–0.85, p = .0003). In the subgroup meta-analyses, this evidence was more consistent among women with Hcy sampling during the second trimester (SMD = 0.76, 95% CI: 0.34–1.18, p = .0004) and with average age ≥30 years (SMD = 0.69, 95% CI: 0.25–1.12, p = .002).Conclusion The evidence indicated that the level of circulating Hcy was significantly elevated among women with GDM compared with those with normal glucose tolerance, especially with mean age ≥30 years and in the second trimester.