Background <p>Observational epidemiology studies suggested a relationship between single-carbon metabolism and vitamin levels with autism spectrum disorder (ASD) risk. We aimed to explore the causal relationship between them at the genetic level.</p> Methods <p>We performed a two-sample bidirectional Mendelian randomization (MR) analysis using genome-wide association studies summary statistics. The inverse-variance weighted (IVW) method was used as the primary analysis. We applied other complementary methods, including weighted median, weighted mode, and MR Egger regression. MR Egger and MR pleiotropy residual sum and outlier (MR-PRESSO) are used to detect and correct the effects of horizontal pleiotropy.</p> Results <p>There were no causal associations between genetically predicted single-carbon metabolism and vitamin levels with ASD risk in IVW analysis (Homocysteine: 95% CI: 0.952–1.082, <i>P</i> = 0.652; Folate in Serum or Plasma: 95% CI: 0.968–1.249, <i>P</i> = 0.143; VB12: 95% CI: 0.989–2.133, <i>P</i> = 0.057; VB6: 95% CI: 0.647–1.247, <i>P</i> = 0.522; VC: 95% CI: 0.794–1.881, <i>P</i> = 0.362; VD: 95% CI: 0.825–1.551, <i>P</i> = 0.443; and VE: 95% CI: 0.87–1.711, <i>P</i> = 0.249). Reversely, we did not find significant causal effects of ASD on single-carbon metabolism and vitamin levels in MR analysis (all <i>P</i> &gt; 0.05). Based on sensitivity analyses, heterogeneity and horizontal pleiotropy are unlikely to distort causal estimates.</p> Conclusions <p>The MR study suggests that no evidence of a causal association was found between single-carbon metabolism and vitamin levels with ASD risk. Further studies are needed to validate these conclusions.</p> Clinical trial number <p>Not applicable.</p>