We investigated serum prolidase activity and oxidative/antioxidative status in patients with knee osteoarthritis (OA) and evaluated its relationships with radiographic severity and clinical parameters. The study population consisted of 137 patients with knee OA and 134 healthy volunteers. The severity of knee OA was classified according to the Kellgren-Lawrence criteria. Each patient was also evaluated clinically according to the Western Ontario and McMaster University Osteoarthritis Index (WOMAC). Serum prolidase activity was measured spectrophotometrically. Oxidative status was assessed by measuring serum lipid hydroperoxide (LOOH) and total oxidative status (TOS). Antioxidative status was assessed by measuring serum-free sulfhydryl groups (-SH = total thiol) and total antioxidant capacity (TAC). Oxidative stress index (OSI) was calculated. Serum prolidase activity was significantly lower in the knee OA group than in the control group (p < 0.001). The serum prolidase activities decreased with the severity of knee OA. Furthermore, serum LOOH, TOS, and OSI levels of the knee OA group were significantly higher than those of the controls (p < 0.001 for all), whereas TAC and -SH levels did not differ between the two groups (p > 0.05). In a multiple regression analysis, WOMAC score was independently associated with serum prolidase activity (beta = -0.340, p < 0.001). Decreased serum prolidase activity and elevated LOOH, TOS, and OSI levels may be associated with knee OA, and serum prolidase activity may be a useful adjunctive indicator of the progression of knee OA in follow-up.