Background: Established risk factors for knee osteoarthritis (KOA) encompass prior joint trauma, advancing age, ethnic background, sex, and the structural configuration of the joint. Oxidative stress and inflammatory processes play pivotal roles in the pathogenesis of the condition. Vitamin D plays a crucial role in upholding skeletal integrity and facilitating the proper metabolism of bone and cartilage. Objective: This study aimed to delve into the nexus between diabetes, oxidative stress, and vitamin D deficiency in individuals afflicted with osteoarthritis. Methods: This study adopted a prospective case-control design involving 90 participants categorized into three equal cohorts: Group 1 comprising OA patients, Group 2 encompassing diabetic patients with osteoarthritis, and Group 3 constituting healthy volunteers. Mild to moderate osteoarthritis Diagnosis adhered to the criteria set forth by the American College of Rheumatology, with participants selected from Outpatient Orthopedic and Rheumatologic Clinics. Serum assessments were conducted across all participants to gauge levels of 25-hydroxyvitamin D (25(OH)D), malondialdehyde (MDA), total oxidant status (TOS), superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT), and total antioxidant capacity (TAC). Results: Significantly disparate findings emerged among the groups in terms of 25(OH)D levels, MDA, TOS, SOD, GPX, CAT, and TAC. Noteworthy associations were delineated between diabetes and both the severity of vitamin D insufficiency and the extent of oxidative stress in osteoarthritis patients. Conclusion: The study discerned an inverse relationship between vitamin D sufficiency and serum concentrations of oxidative stress markers, including MDA, TOS, SOD, and OSI. Furthermore, a positive correlation was unveiled between TAC and serum vitamin D levels.