Aerobic exercise is recommended to alleviate pain and protect the joint for patients with advanced knee osteoarthritis, however, its clinical implementation is challenging due to the potential for exacerbating pain. The study aimed to compare the effects of anti-gravity treadmill training with traditional treadmill training in patients with advanced knee osteoarthritis. This single-blinded randomized-controlled trial included 30 women with knee osteoarthritis. All participants received hotpack, transcutaneous electrical nerve stimulation, and therapeutic ultrasound. Additionally, group 1 received anti-gravity treadmill, while group 2 received traditional treadmill training. Group 3 served as the control. The interventions were administered three-times a week for eight-weeks. The visual analogue scale (VAS) pain, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), six-minute-walk-test distance (6MWD), and femoral cartilage thickness were evaluated at baseline and weeks 4 and 8. VAS-pain significantly reduced over time in both anti-gravity (P < 0.001) and control (P = 0.004) groups. The anti-gravity group also showed significant improvements in WOMAC-pain (P = 0.008), WOMAC-total (P = 0.048), and 6MWD (P < 0.001). Post-hoc analysis indicated significant time (P < 0.001, effect size, ηp2 = 0.682) and interaction (P = 0.006, ηp2 = 0.271) effects on VAS, with no significant between-group differences. Femoral cartilage thickness showed no significant between-group differences, except within-group differences in the treadmill group (P = 0.037). Anti-gravity treadmill training significantly improved pain, functionality, and functional capacity in patients with knee osteoarthritis, while traditional treadmill resulted in a reduction in femoral cartilage thickness. Further research should investigate long-term outcomes and more diverse populations. NCT05319964.