Objective: The association of diabetes mellitus (DM) with increased risk of knee osteoarthritis (OA) is uncertain. We evaluated associations of DM and biomarkers of abnormal glucose metabolism with incident radiographic knee OA, controlling for body mass index (BMI). Methods: Participants (mean ± SD age 60.6 ± 7.8 years; mean ± SD body mass index [BMI] 29.1 ± 4.9 kg/m2) were from the Multicenter Osteoarthritis Study and did not have radiographic knee OA at baseline (Kellgren/Lawrence [K/L] grade <2 bilaterally). A random sample (n = 987) was selected and stratified by BMI. Baseline serum fasting glucose and homeostasis model assessment–estimated insulin resistance (HOMA-IR) were measured. Participants were categorized as having DM based on self-report, use of medication, or fasting glucose ≥126 mg/dl. Incident radiographic knee OA (K/L grade ≥2 or knee replacement) was assessed at 3 follow-up visits (30, 60, and 84 months). Knee-level pooled logistic regression analysis was performed to obtain odds ratios (ORs) (95% confidence interval [95% CI]) for associations of DM status and biomarkers of abnormal glucose metabolism with incident radiographic knee OA. Results: After adjustment for BMI, the odds of incident radiographic knee OA were not associated with baseline DM status nor with levels of fasting glucose and HOMA-IR, overall and in men. In women, HOMA-IR was inversely associated with odds of incident radiographic knee OA (adjusted OR 0.80 [95% CI 0.69–0.94], P = 0.005). Conclusion: DM and higher levels of biomarkers of abnormal glucose metabolism were not associated with increased odds of incident radiographic knee OA after adjusting for BMI in this cohort overall. A possible protective association of higher HOMA-IR with incident radiographic knee OA in women warrants further investigation.