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Background: The role of prolotherapy in the healing process of damaged tissues has been confirmed in patients with degenerative joint disease. Hypertonic dextrose has been introduced as the most common solution in prolotherapy for reducing pain and improving mobility in people with knee osteoarthritis (KOA). This study aimed to compare the KOA patients undergoing hypertonic dextrose prolotherapy (DPT) with the placebo in terms of pain intensity and functional level. Methods: This triple-blind study randomized clinical trial (RCT) was conducted on patients with mild to moderate KOA who were referred to the Rheumatic Diseases Research Center (RDRC), Mashhad University of Medical Sciences, Mashhad, Iran, in the year 2021. Patients were randomly assigned to two groups of the DPT (n = 47) and normal saline placebo prolotherapy (n = 50) intra- and peri-articular knee joints. Treatment was monthly for three months. Western Ontario and McMaster Universities’ Arthritis Index (WOMAC) and Anterior Knee Pain Scale (AKPS) were completed before and after intervention (weeks 0, 4, 8, 12, and 16). The variables were compared between the two study groups at a significance level of less than 0.05 (P < 0.05). Results: The WOMAC score improved significantly compared to the baseline in both groups (P < 0.005). An increase in WOMAC score (the last follow-up compared to baseline) was 97.4% and 85.7% in DPT and placebo groups, respectively (P = 0.06). Before the intervention and also after the first injection, the AKPS score in the normal saline group was significantly better than the dextrose group (P = 0.03). The AKPS score did not differ significantly between the two groups (P > 0.05) in the next two visits and two-month follow-ups. Improvement of AKPS score in the last follow-up compared to baseline was reported in 66.7% and 68.4% of patients in the normal saline group and dextrose group, respectively, (P = 0.86). Conclusion: Prolotherapy as intra- and peri-articular injections of hypertonic dextrose could be used to relieve pain and improve functional status in patients with mild to moderate KOA as much as a placebo does. Although no remarkable difference was observed between dextrose and placebo in the outcomes, the rate of improvement (last follow-up compared to the baseline) was relatively higher in the dextrose group than in the normal saline group. [ABSTRACT FROM AUTHOR]