Multiple sclerosis (MS) is a prevalent neurological disorder with a complex etiology, often associated with thyroid function. However, the causal relationship between these two conditions remains poorly understood. This study aimed to elucidate the causal relationship between thyroid function and MS using a bidirectional Mendelian randomization (MR) approach and to investigate the potential mediating role of immune cells. We conducted a two-sample MR analysis using summary statistics from large-scale genome-wide association studies (GWAS). We included results from sensitivity tests such as MR-Egger, weighted median, and leave-one-out analyses to support the robustness and reliability of the findings. The inverse variance-weighted (IVW) method was the primary approach, with sensitivity analyses conducted using seven additional MR methods. Furthermore, multivariable MR and mediation analysis were conducted to uncover potential mediating immune cells underlying the observed associations. The MR analysis showed that Hypothyroidism and elevated Thyroid-Stimulating Hormone (TSH) levels(normal) reduced the risk of MS (P = 0.012, OR (95%CI) :0.914(0.851, 0.98)
P = 0.020, OR (95%CI) :0.88(0.789, 0.98)). Free thyroxine (FT4) increased the risk of MS (P = 0.020, OR (95%CI) :1.268(1.051, 1.53)). Mediation analysis showed evidence of indirect effect of FT4 on MS through "HLA DR on CD33br HLA DR + CD14" and "IgD- CD27- %B cell" with a mediated proportion of 39.16% (positive effect), 78.53% (reverse effect) of the total effect. This study provided genetic evidence that FT4 may increase the risk of developing MS. "HLA DR on CD33br HLA DR + CD14" and "IgD- CD27- %B cell", mediated the causal relationship between thyroid function and MS, highlighting the importance of further investigating their roles in these conditions.