PURPOSE: To investigate the effects of an 8-week proprioceptive neuromuscular facilitation (PNF) intervention on pain and biomechanical characteristics during stepping over obstacles among older adults with Knee Osteoarthritis (KOA). METHODS: Thirty-two participants were randomly allocated into PNF or control groups. The participants in the PNF group received PNF stretching and those in the control group partook in the health lecture series for eight weeks, with three one-hour sessions a week. The Visual Analogue Scale (VAS) was used to assess the pain score. Each subject stepped over an obstacle with a height of 20% leg length. The spatiotemporal variables of the more affected leg were collected using a motion analysis system with two force platforms before and after the intervention. Joint angles and moments were calculated. Two-way (group by intervention) ANOVA with repeated measures was used to compare the differences. RESULTS: Data analysis included 13 participants in the PNF group (8 female, 65.77 ± 4.69 years, 165.46 ± 6.78 cm, and 71.53 ± 8.89 kg) and 14 in the control group (7 female, 63.00 ± 2.77 years, 161.41 ± 6.75 cm, and 69.06 ± 9.59 kg). Significant interactions were detected in the following variables, the pain score (week0: 4.30 ± 1.45, week9: 1.32 ± 1.25, p < 0.001, d = 2.189), foot clearance (week0: 0.27 ± 0.04, week9: 0.25 ± 0.03, p = 0.036, d = 0.555), knee external rotation (week0: 16.14 ± 7.80, week9: 11.08 ± 7.86, p = 0.017, d = 0.646) angles, and first (week0: 0.40 ± 0.10, week9: 0.24 ± 0.10, p < 0.001, d = 1.583) and second peaks (week0: 0.36 ± 0.12, week9: 0.17 ± 0.12, p < 0.001, d = 1.583) of the knee adduction moment were significantly decreased, while the crossing velocity (week0: 0.30 ± 0.04, week9: 0.33 ± 0.03, p = 0.003, d = 0.832), hip flexion angle (week0: 76.16 ± 6.94, week9: 82.51 ± 5.75, p = 0.004, d = 0.996), hip flexion (week0: 0.79 ± 0.23, week9: 0.95 ± 0.17, p = 0.007, d = 0.774) and extension (week0: 0.49 ± 0.09, week9: 0.61 ± 0.14, p < 0.001, d = 0.977) moments were increased after the intervention in PNF group, but not in the control group. CONCLUSION: PNF could relieve pain, improve dynamic joint flexibility and gait strategies among older adults with knee osteoarthritis during stepping over obstacles. Funding was provided by Shandong Province Young Innovative Talent Introduction and Cultivation Program (2019-183).