Increasing evidence suggests that the status of vitamin D and n-3 PUFA is associated with the risk of CVD. Major dietary sources of vitamin D include fish and fish products, which are also rich in n-3 PUFA; however, the relationship between serum 25-hydroxyvitamin D levels and tissue contents of n-3 PUFA remains unknown. The present study investigates the hypothesis that serum 25-hydroxyvitamin D and erythrocyte n-3 PUFA levels are positively correlated in patients with CVD. We recruited sixty CVD cases and matched them with sixty healthy controls based on age, sex and season during which blood was drawn for the study. As serum 25-hydroxyvitamin D levels increased, erythrocyte levels of docosapentaenoic acid, DHA, omega-3 index and total n-3 PUFA increased significantly, while erythrocyte levels of stearic acid and total SFA decreased significantly, after adjusting for age, sex, BMI and smoking. Partial correlation analysis also showed that erythrocyte n-3 PUFA levels were positively correlated (r 0·215; P = 0·021) and total SFA content was negatively correlated (r − 0·263; P = 0·004) with serum 25-hydroxyvitamin D levels. However, multiple logistic regression analysis showed that serum 25-hydroxyvitamin D levels were not significantly associated with the risk of CVD, after adjusting or not adjusting for age, sex, BMI and smoking. In conclusion, the results of our case–control study suggest that serum 25-hydroxyvitamin D levels are positively related to erythrocyte n-3 PUFA levels, but are not associated with the risk of CVD in this population.