Objective <p>This study investigates the safety and efficacy of three intra-articular (IA) injections of cryopreserved human placenta-derived mesenchymal stem cells (hP-MSCs) for knee osteoarthritis (KOA) over a 1-year follow-up period.</p> Methods <p>A total of 26 patients with stage II-III KOA were enrolled in this non-randomized, open-label study. Patients received either conventional therapy with hyaluronic acid (HA) alone (Control group, <i>n</i> = 11) or in combination with hP-MSCs (MSC group, <i>n</i> = 15) via three intra-articular injections with 4-week intervals. Clinical outcomes were assessed using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Visual Analogue Scale (VAS), and magnetic resonance imaging (MRI) at 6 and 12 months following the first injection. Blood samples were analyzed for cytokine levels.</p> Results <p>Three injections of hP-MSCs combined with HA were well-tolerated, with no severe adverse events observed. Significant improvements in WOMAC and VAS scores were noted in the MSC group compared to the Control group at both 6 and 12 months. MRI analysis revealed no significant differences in cartilage thickness or optical density index between the groups. Additionally, serum cytokine analysis showed a significant decrease in interleukin-2 (IL-2) levels in the MSC group, indicating an anti-inflammatory effect of hP-MSCs. However, no significant changes were observed in other cytokines.</p> Conclusion <p>This study demonstrates that three intra-articular injections of cryopreserved hP-MSCs in combination with HA are safe and effective for treating KOA, providing sustained clinical improvement at the 1-year follow-up.</p> Trial registration <p>NCT04453111, #7/09.26.2018. Registered 02 January 2020, <a href="https://clinicaltrials.gov/study/NCT04453111">https://www.clinicaltrials.gov/study/NCT04453111</a>.</p>