The study aims to identify clinical factors affecting tacrolimus blood trough concentration (C0) in myasthenia gravis (MG) patients and to optimize the initial dose of tacrolimus in MG treatment. A total of 103 MG patients participated in this study, and their clinical factors, medication regimens, C0 values and CYP3A5*3 polymorphisms were collected in detail. We used a linear mixed model to analyze the effect of multiple factors on the dosage-weighted C0 (C0:D) and performed subgroup analyses to investigate the consistency of correlations between influencing factors and the C0:D ratios. Among all factors, CYP3A5*3 polymorphism and age showed a strong positive correlation with C0:D ratios. The C0:D ratios (ng/ml·mg−1) were higher for CYP3A5*3/*3 than for CYP3A5*1 (mean difference: 1.038, 95% confidence interval [CI]: 0.820–1.256, P-value <0.001), and for age in the range of 45–64 and ≥ 65 years than for age < 45 years (mean difference [95% CI] and P-value: 0.531[0.257–0.805] and P-value <0.001, 0.703 [0.377–1.029] and P-value <0.001, respectively). The C0:D ratios were not related to corticosteroid dosage, body weight, sex, hematocrit or the concomitant use of calcium channel blockers. The consistencies of the correlations between C0:D ratios and CYP3A5*3 polymorphism or age were confirmed by subgroup analyses. Thus, CYP3A5*3 polymorphism and age should be considered in optimizing the initial dose of tacrolimus for MG treatment. © 2021 Elsevier B.V.