Limited research has examined the association between diabetes mellitus (DM) and knee pain in people with osteoarthritis (OA). Therefore, this study aimed at examining the association between DM and knee pain severity, and to explore the association between DM and knee pain distribution (unilateral or bilateral versus no pain) in subjects with knee OA. This is a cross-sectional analysis of the baseline visit of individuals who were enrolled in the Osteoarthritis Initiative. Data of participants with knee OA were used for this analysis (n = 1319), and grouped into subjects with both knee OA and DM (n = 148) or knee OA only without DM (n = 1171). Pain severity was measured using a numeric rating scale from 0 to 10 over the past 7 and 30 days for each knee, and the more symptomatic knee with higher pain severity was chosen for analysis. DM was significantly associated with increased knee pain severity over 7 days (B 0.68; 95% CI 0.25-1.11) and over 30 days (B 0.59; 95% CI 0.17-1.01) after adjustments for all covariates, including age, gender, BMI, race, depression symptoms, composite OA score, use of pain medications, and knee injections. Multinomial regression showed that participants with knee OA and DM had 2.45 (95% CI 1.07-5.61) to 2.55 (95% CI 1.12-5.79) times higher likelihood of having unilateral and bilateral knee pain than those without DM and without knee pain. This study found that DM was associated with higher pain severity and unilateral and bilateral knee pain distribution.