Abstract Purpose Low serum vitamin D status has been associated with reduced muscle mass in observational studies although the relationship is controversial and a causal association cannot be determined from such observations. Two-sample Mendelian randomization (MR) was applied to assess the association between serum vitamin D (25(OH)D) and total, trunk, arm and leg fat-free mass (FFM). Methods MR was implemented using summary-level data from the largest genome-wide association studies (GWAS) on vitamin D (n=73,699) and total, trunk, arm and leg FFM. Inverse variance weighted method (IVW) was used to estimate the causal estimates. Weighted median (WM)-based method, and MR-Egger, leave-one-out were applied as sensitivity analysis. Results Genetically higher serum 25(OH)D levels had a positive effect on total (IVW = Beta: 0.042, p = 0.038), trunk (IVW = Beta: 0.045, p = 0.023) and arm (right arm IVW = Beta: 0.044, p = 0.002; left arm IVW = Beta: 0.05, p = 0.005) FFM. However, the association with leg FFM was not significant (right leg IVW = Beta: 0.03, p = 0.238; left leg IVW = Beta: 0.039, p = 0.100). The likelihood of heterogeneity and pleiotropy was determined to be low (statistically non-significant), and the observed associations were not driven by single SNPs. Furthermore, MR pleiotropy residual sum and outlier test did not highlight any outliers. Conclusions Our results illustrate the potentially causal, positive effect of serum 25(OH)D concentration on total, trunk and upper body appendicular fat-free mass.