Background: Anemia is a common disease in middle-aged and elderly people, and the occurrence of anemia greatly reduces the quality of life of this population. Middle-aged and elderly people often feel weakness and fatigue associated with anemia, 25-hydroxyvitamin D is thought to have an effect on erythropoiesis, and there is limited research on the relationship between anemia and deficiency of 25-hydroxyvitamin D deficiency; therefore, research is needed to investigate the relationship between anemia and deficiency of 25-hydroxyvitamin D. Objective: To assess the association between 25-hydroxyvitamin D levels and anaemia in middle-aged and older adults. Methods: This cross-sectional study was conducted from January 2021 to December 2022, and a total of 5320 patients were examined at the First People's Hospital of Anning City, Yunnan Province. Serum 25-hydroxyvitamin D levels and blood cell parameters were also measured., and the relationship between serum 25-hydroxyvitamin D levels and these parameters was analyzed using Pearson correlation analysis and a generalized additive model with a smoothing function. Results: The average serum 25-hydroxyvitamin D level in the middle-aged and elderly population was 21.39 ng/ml, and only 12% of individuals had normal 25-hydroxyvitamin D levels (>= 30 ng/ml).The level of 25-hydroxyvitamin D was positively and nonlinearly with the number of red blood cells and hemoglobin content (P < 0.01). Conclusion: Vitamin D deficiency or insufficiency is common in middle-aged and elderly individuals. Serum 25-hydroxyvitamin D levels can affect the levels of red blood cells and hemoglobin. Close monitoring and appropriate correction of serum vitamin D levels may improve anemia in clinical practice.