Several studies have reported improvement in knee pain following mesenchymal stem cell (MSC) injections for knee osteoarthritis (OA). We developed a novel 3D magnetic resonance imaging (MRI) analysis software program that provides "projected cartilage area ratios" for automatic detection of changes in cartilage amounts. The primary objective of this prospective interventional study was to compare alterations in the projected cartilage area ratio (thickness ≥ 1.5 mm) at the femoral posteromedial region between 30 weeks before and 30 weeks after synovial MSC injections. Secondary objectives were to assess the clinical scores and safety of MSC injections. Patients with OA who complained of knee pain underwent autologous synovial MSC injections into the knee at time 0 and again 15 weeks later. MRI examinations were performed at - 30, - 15, - 1, and 30 weeks. Patients showing < 3% decreases in the projected cartilage area ratio (thickness ≥ 1.5 mm) at the femoral the posteromedial region from - 30 weeks to - 15 weeks were excluded from the study. The Lysholm Knee Score, Knee Injury and Osteoarthritis Outcome Scale (KOOS), and Numerical Rating Scale (NRS) scores were evaluated at - 30, - 15, - 5, - 2, 0, 5, 10, 15, 20, 25, and 30 weeks. Five patients were excluded because 3D MRI analysis showed no cartilage loss at - 15 weeks. Ultimately, eight OA patients underwent MSC injections. The projected cartilage area ratio significantly decreased by 0.07 in the 30 weeks before MSC injections (p = 0.01), but no further decreases occurred in the 30 weeks after MSC injections. The projected cartilage area ratio at the femoral posteromedial region showed a significant difference between 30 weeks before and 30 weeks after MSC injections. The Lysholm Knee Score, KOOS, and NRS values improved significantly after the injections. MSC injection could not be ruled out as the cause of two adverse events: transient knee pain and itching in both hands. Fully automatic 3D MRI analysis showed that synovial MSC injections suppressed cartilage loss in patients with progressive OA.Trial registration: Intraarticular injections of synovial stem cells for osteoarthritis of the knee (Number UMIN 000026732). Date of registration; June 1, 2017. https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000029967 .