This randomized, double-blind clinical trial was conducted over 16 weeks to assess the efficacy and safety profile of intra-articular polynucleotides gel injections in the treatment of knee osteoarthritis associated with persistent knee pain. 60 patients were enrolled and randomized to receive intra-articular polynucleotides (n = 30) or hyaluronan (n = 30); patients received five weekly intra-articular knee injections and the follow-up period was 3 months after the end of treatment. Primary endpoint was to determine polynucleotides (PN) efficacy in reducing knee pain at the end of the study, over baseline value and over standard hyaluronan viscosupplementation (HA). Pain levels were measured using a 0-10 cm Visual Analogue Scale (VAS). Secondary endpoints included Knee Osteoarthritis Outcome Score (KOOS), NSAIDs consumption, crackling during movement and articular mobility limitation. The mean global VAS pain decreased from 5.7 + or - 1.9 cm (T0) to 1.9 + or - 1.5 cm (T16) in polynucleotide group and from 4.9 + or - 2.0 cm (T0) to 2.1 + or - 1.4 cm (T16) in hyaluronan group. The reduction in pain was statistically significant for both groups. KOOS increases from baseline values were statistically significant in both groups. No significant adverse events were reported. These findings suggest that intra-articular polynucleotides can be a valid alternative to traditional hyaluronan supplementation for the treatment of knee osteoarthritis.