Objectives <p>To evaluate the safety and clinical efficacy of intra-articular injection of human umbilical cord–derived mesenchymal stem cells (UC-MSCs) for knee osteoarthritis (KOA).</p> Methods <p>This was a randomized, double-blind, placebo-control trial consisting of 3 arms including placebo, hyaluronic acid (HA), and UC-MSCs. 69 individuals were screened, with 55 participants subsequently randomized. The primary endpoint was the safety after injection. The secondary endpoints were improvements in visual analog scale (VAS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Knee Society Clinical Rating System (KSS), EuroQol five-dimensional questionnaire (EQ-5D), and magnetic resonance imaging (MRI) examinations at 6-month after injection.</p> Results <p>Adverse events (AEs) did not demonstrate significant differences among the groups. No serious treatment-related AEs were observed. Compared with placebo and HA, injection of UC-MSCs resulted in a greater improvement in VAS and EQ-5D; however, the differences were not statistically significant. Patients in the UC-MSCs group exhibited significant improvement in synovitis at the 6-month follow-up as compared with the placebo and HA groups (P = 0.003). At 6 months, UC-MSCs injection showed significantly better improvements in VAS (P = 0.011), WOMAC (P = 0.014), and EQ-5D scores (P = 0.049) in patients with KOA incorporating synovitis, particularly those experiencing moderate to severe pain. MRI indicated no significant difference in change of joint structures among the groups, except for the synovium.</p> Conclusions <p>Treatment with UC-MSCs was shown to be a viable therapeutic option for KOA combined with synovitis, showing clinical improvement at the end of follow-up, especially in those with moderate to severe pain. Further phase III clinical trials would be required to confirm the efficacy.</p> Trial registration <p>The trial was registered at chictr.org on October 13, 2020, with the registration number ChiCTR2000039017.</p>