Introduction: Chronic Kidney Disease (CKD) is a major global health issue, with an increasing prevalence in Bangladesh. It is associated with numerous complications, especially cardiovascular diseases, which are the leading cause of morbidity and mortality among CKD patients. Arterial stiffness, a key cardiovascular risk factor, has been shown to correlate with CKD progression. Recent studies suggest that serum 25-hydroxyvitamin D (25(OH)D) deficiency plays a significant role in the development of arterial stiffness and cardiovascular complications in CKD patients. Objective: This study aims to investigate the impact of serum 25-hydroxyvitamin D deficiency on arterial stiffness in non-dialysis CKD patients in Bangladesh. Methods: A cross-sectional observational study was conducted at the Department of Nephrology, Dhaka Medical College, between September 2021 and March 2023. A total of 100 non-dialysis CKD patients (Stages 3-5) were enrolled, with 50 patients each in the vitamin D deficient and non-deficient groups. Arterial stiffness was measured using established methods, and serum 25(OH)D levels were assessed. Statistical analysis was performed using SPSS version 26.0, with significance set at p<0.05. Results: The study found that 78% of patients in the vitamin D deficient group were in Stage 5 CKD, compared to 12% in the non-deficient group. A significant negative correlation was observed between serum 25(OH)D levels and arterial stiffness (r = -0.386, p = 0.001). Laboratory variables such as eGFR and hemoglobin levels were significantly lower in the vitamin D deficient group (p<0.05). Conclusion: The study concludes that vitamin D deficiency plays a critical role in the pathogenesis of arterial stiffness in CKD patients. Addressing vitamin D deficiency may offer potential benefits in mitigating cardiovascular complications and improving the prognosis of CKD patients in Bangladesh.