Background and Aims. The effects of cortisol on cardiovascular diseases (CVD) and CVD risk are unknown, especially in patients with type 2 diabetes mellitus (T2DM). Furthermore, it is unclear whether 25 (OH)D can alter the associations of cortisol with CVD and CVD risk factors. Thus, the present study was to investigate the associations of serum cortisol with CVD and CVD risk factors and whether 25 (OH)D altered these associations among patients with T2DM. Materials and methods. A total of 762 patients diagnosed with T2DM were recruited. The levels of serum cortisol and 25 (OH)D were measured with a liquid chromatography-tandem mass spectrometry. Logistic regression and linear regression were used to assess the association of cortisol with CVD and multiple cardiovascular risk factors. Modification analyses were performed to identify whether 25 (OH)D altered the above associations. Results. A 1 SD increase in cortisol was associated with a higher prevalence of stroke (odds ratio (OR): 1.25, 95% confidence interval (CI): 1.05, 1.50). Elevated cortisol was associated with related cardiovascular risk factors, including deceased ß cell function, high-density lipoprotein-cholesterol (HDL-C), and fasting insulin, as well as increased triglycerides (TG), low-density lipoprotein-cholesterol (LDL-C), fasting plasma glucose (FPG), and glycated hemoglobin (HbA1c). In addition, modification analyses suggested that the associations of cortisol with ß cell function, fasting insulin, FPG, and HbA1c were modified by 25 (OH)D. Conclusions. Serum cortisol was associated with the prevalence of stroke and cardiovascular risk factors, and the associations of cortisol with cardiovascular risk factors were moderated by 25 (OH)D, suggesting that T2DM patients with exposure to lower 25 (OH)D levels and higher cortisol levels were more susceptible to have higher cardiovascular risk factors.