Limited studies have shown that exposure to arsenic is associated with serum vitamin D levels, but the results are still inconsistent. A cross-sectional study of 762 participants was conducted in Wenshui Country, Shanxi Province, which was identified as an area of water-borne arsenicosis. The results showed positive relationship between arsenic species (iAs, MMA, DMA) and serum 25(OH)D. Binary logistic regression analysis showed that the significant increases of 0.4% and 0.6% in the risk of vitamin D excess for every 1-unit increment in the Box-Cox transformed urinary tAs and DMA, respectively. After stratifying populations based on arsenic methylation metabolic capacity, each one-unit increase in the Box-Cox transformed tAs level was associated with increases of 0.064 (95%CI: 0.032 to 0.096) in serum 25(OH)D in the populations with iAs% above the median. In the populations with skin hyperkeratosis, urinary iAs was positively associated with serum 25(OH)D (β=0.592, 95%CI: 0.041 to 1.143). Overall, our findings support the positive relationship between urinary arsenic and serum 25(OH)D.