Although the use of intra-articular polynucleotide (IA PN) injection as a viscosupplement for knee osteoarthritis (OA) treatment has been proposed, its efficacy and safety compared to high molecular weight hyaluronic acid (HMWHA) injection has not yet been established. The present double-blind, multicenter, randomized controlled trial aimed to investigate the efficacy and safety of IA PN injection compared to IA HMWHA injection. A total of 60 patients (15 men, 45 women, 64.5 ± 7.5 years) with knee OA (Kellgren–Lawrence grade 1–4) were randomly allocated to each group. All patients were given three IA injections of PN (n = 30) or HMWHA (n = 30) at intervals of 1 week. The primary endpoint was the change rate in weight-bearing pain (WBP) 16 weeks from the baseline. The secondary endpoint included multiple measurements: the change rate in WBP rate at 8 weeks; the change rate in pain level at rest and during walking at 8 and 16 weeks; the Korean-Western Ontario and McMaster University Osteoarthritis index; the Euro-Quality of Life-5 Dimension; Clinical Global Impression, Patient Global Impression at 8 and16 weeks, and total consumption of rescue medicine. The mean change rate in the WBP at 16 weeks from the baseline was − 54.0 ± 38.1% in the IA PN group and − 42.8 (± 35.8%) in the IA HMWHA group, and there was no significant difference between the two groups (p= 0.296). All secondary endpoints related with pain and functional outcome also showed no significant difference between the two groups. Pain at the injection site and swelling were reported as adverse events, and the incidence was similar between the two groups. IA PN showed comparable efficacy and safety to IA HMWHA at 3 times injection with an interval of 1 week. IA PN can be useful alternative to IA HMWHA for the treatment of knee OA.