This study was designed to explore the mediating role of serum 25-hydroxyvitamin D (25(OH) D) in triglyceride glucose (TyG) index and hypertension (HTN). Study participants were selected from the 2001-2018 National Health and Nutrition Examination Survey. Firstly, we estimated the association between TyG index, and serum 25(OH)D with HTN using weighted multivariable logistic regression model and restricted cubic spline. Secondly, we used generalized additive model to investigate the correlation between TyG index and serum 25(OH)D. Lastly, serum 25(OH)D was investigated as a mediator in the association between TyG index and HTN. There were 14,099 subjects in total. TyG index was positively and linearly associated with HTN risk, while serum 25(OH)D had a U-shaped relationship with prevalence of HTN. When the serum 25(OH)D levels were lower than 57.464 mmol/L, prevalence of HTN decreased with the increase of serum 25(OH)D levels. When serum 25(OH)D levels rise above 57.464 mmol/L, risk of HTN increased rapidly. Based on the U-shaped curve, serum 25(OH)D concentrations were divided into two groups: (<57.464 and ≥57.464 mmol/L). According to the mediation analysis, when serum 25(OH)D levels <57.464 mmol/L, the positive association between TyG index and incident HTN was increased by 25(OH)D. When serum 25(OH)D levels ≥57.464 mmol/L, the negative association between TyG index and incident HTN was increased by 25(OH)D. There was a mediation effect between TyG index and HTN, which was mediated by 25(OH)D. Therefore, we found that the association between serum 25(OH)D levels and TyG index may influence prevalence of HTN.