Background: High-intensity laser therapy (HILT) is still a controversial physical therapy modality for individuals with knee osteoarthritis (OA). Objectives: This study aimed to evaluate the effect of HILT on pain and function in patients with knee OA. Methods: This randomized controlled trial included 56 patients with knee OA referred to Amin Hospital affiliated with Isfahan University of Medical Sciences, Isfahan, Iran, from April 19 to August 30, 2022. Patients were randomized into two groups (HILT and control), receiving isometric exercises targeting the quadriceps muscle, meloxicam 15 mg tablet, and Rahamin ointment for two weeks. The HILT group also received high-intensity laser irradiation three times a week for two weeks. The primary outcome was pain assessed using a Visual Analog Scale (VAS), and the secondary outcome was the Western Ontario and McMaster Universities Osteoarthritis index (WOMAC) and its subscales (pain, stiffness, and physical function). Outcomes were evaluated at baseline, immediately, and three months after the last treatment session. Results: After adjusting for age, sex, and baseline values, immediately and three months after treatment, VAS pain scores were significantly lower in the HILT group than in controls (P < 0.001), with a large effect (standardized mean difference (SMD) = -1.22, 95% confidence interval (CI): -1.81; -0.62 and SMD = -1.31, 95% CI: -1.90; -0.70, respectively). The results were similar for WOMAC's pain subscale. Despite the difference in WOMAC stiffness score was not significant between the two groups immediately after treatment (P = 0.135), this score was significantly lower in the HILT group than in the control group (SMD = -0.98, 95% CI: -1.56; -0.40, P = 0.002) three months after treatment. Furthermore, WOMAC physical function scores were significantly lower with HILT immediately and three months after treatment (P < 0.001). The same was true for the total WOMAC score. Conclusions: High-intensity laser therapy significantly improved pain and the WOMAC score in patients with knee OA immediately and three months after the last treatment session compared to the control group.