Background: Mesenchymal stromal cells (MSCs) injection has been proposed as an innovative treatment for knee osteoarthritis (KOA). Since, allogeneic MSCs can be available as off-the-shelf products, they are preferable in regenerative medicine. Among different sources for MSCs, adipose-derived MSCs (AD-MSCs) appear to be more available. Methods: Three patients with KOA were enrolled in this study. A total number of 100 × 106 ADMSCs was injected intra-articularly, per affected knee. They were followed up for 6 months by the assessment of clinical outcomes, magnetic resonance imaging (MRI), and serum inflammatory biomarkers. Results: The primary outcome of this study was safety and feasibility of allogeneic AD-MSCs injection during the 6 months follow-up. Fortunately, no serious adverse events (SAEs) were reported. Assessment of secondary outcomes of visual analogue scale (VAS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and knee osteoarthritis outcome score (KOOS) indicated improvement in all patients. Comparison between baseline and endpoint findings of MRI demonstrated a slight improvement in two patients. In addition, decrease in serum cartilage oligomeric matrix protein (COMP) and hyaluronic acid (HA) indicated the possibility of reduced cartilage degeneration. Moreover, quantification of serum interleukin-10 (IL-10) and interleukin-6 (IL-6) levels indicated that the host immune system immunomodulated after infusion of AD-MSCs. Conclusion: Intra-articular injection of AD-MSCs is safe and could be effective in cartilage regeneration in KOA. Preliminary assessment after six-month follow-up suggests the potential efficacy of this intervention which would need to be confirmed in randomized controlled trials on a larger population. Trial registration: This study was registered in the Iranian registry of clinical trials (https://en.irct. ir/trial/46) in 24 April 2018 with identifier IRCT20080728001031N23.