To assess the efficacy of a 12-week aquatic cycling training program for improving knee pain and physical functioning in patients with knee osteoarthritis (OA). Two-arm, single-blind, parallel-group randomized controlled trial. OA outpatient clinic of the Maastricht University Medical Center+. Patients (N=111, 50-70y) with unilateral mild-to-moderate knee OA. Participants (aquatic cycling [AC] group, n=55) received AC sessions of 45 min each 2 times per week. Each session combined upright seated cycling with out-of-saddle positions and exercises for the upper and lower body. The usual care (UC) group (n=47) continued with UC and was offered 12 AC sessions in a local swimming pool after their trial participation. The Knee Injury and Osteoarthritis Outcome Score (KOOS) on knee pain and physical function was assessed at baseline, postintervention, and at 24-wk follow-up. Multilevel (mixed regression) analysis examined the effects. Average attendance rate for the AC sessions was 80%. Statistically significant differences at postintervention and follow-up were found for knee pain in mean ± SD (UC pretest, 57.89±15.26; posttest, 55.90±18.04; follow-up, 57.24±19.16; and AC pretest, 56.96±12.96; posttest, 63.55±15.33; follow-up, 64.35±17.26; estimate, 8.16; SE, 3.27; 95% confidence interval [CI], 1.67-14.64; effect size [ES], 0.50) and physical functioning (UC pretest, 66.32±16.28; posttest, 66.80±19.04; follow-up, 65.42±17.98; and AC pretest, 61.89±17.151; posttest, 70.14±17.52; follow-up, 69.00±16.84; estimate, 7.16; SE, 3.19; 95% CI, 0.83-13.49; ES, 0.43) in favor of the aquatic group. The results suggest that a 12-week AC training program improves self-reported knee pain and physical functioning in patients with mild-to-moderate knee OA compared to UC.