Objective To explore the association between cystatin C (CysC) and Hashimoto thyroiditis (HT), and to clarify the potential role of CysC in HT. Methods The newly diagnosed HT patients admitted to the Affiliated Hospital of Jiangsu University between January 2022 and December 2023 were selected as HT group and the same number of healthy physical examinees were selected as control group. The serum CysC levels of the two groups were compared. Subsequently, Mendelian randomization (MR) method (inverse variance weighted) was used to explore the association between CysC and HT. Results A total of 30 HT group patients and 30 healthy people were included. There was no significant difference in age and female proportion between HT group and control group (P>0.05). The serum CysC level in the HT group was higher than that in the control group [(0.91±0.16) vs. (0.79±0.11) mg/L, P<0.05]. MR analysis showed that higher serum CysC level would increase the risk of HT (P<0.05). The results of reverse MR analysis showed that there was no causal relationship between HT as an exposure factor and CysC as an outcome (P>0.05). ConclusionClinical serological test results and MR analysis suggested that CysC may play an important role in the pathogenesis of HT.