Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) are a potential off-the-shelf product for acute ischemic stroke. This study explored the underlying mechanism of Cytopeutics® hUC-MSCs (Neuroncell-EX) as well as its feasibility and efficacy at two different doses: 2 × 10 cells per rat and 4 × 10 cells/rat in middle cerebral artery occlusion (MCAO) ischemic stroke model for 28 d. Modified neurological severity score (mNSS) and rotarod tests were evaluated at days 1, 4, 7, and 14. Transforming growth factor-beta 1 (TGF-β1), interleukin-1 receptor antagonist (IL-1Ra), and vascular endothelial growth factor (VEGF) were evaluated by enzyme-linked immunosorbent assay (ELISA) at days 4 and 28. Immunohistochemistry expression of aquaporin-4 (AQP4) and neuronal protein marker (NeuN) were performed at days 4 and 28, respectively. Both doses of Neuroncell-EX showed significant lower mNSS scores at days 7 and 14 compared to stroke control. Both Neuroncell-EX groups showed significant longer latency time at day 7, with only 4 × 10⁶ cells/rat group having significant longer time at day 14 than stroke control. At both time points, the 2 × 10⁶ cells/rat group had significantly higher TGF-β1 and IL-1Ra levels, with significantly increased TGF-β1 only observed in 4 × 10⁶ cells/rat group at day 4 compared to stroke control. The VEGF levels were significantly lower at day 4 but then significantly increased at day 28 in both Neuroncell-EX groups than stroke control. AQP4 expression was significantly higher in stroke control compared to healthy control at day 4. Both doses of Neuroncell-EX showed significantly higher NeuN expression compared to stroke control at day 28. There is a weak correlation between TGF-β1 with VEGF and inversely with AQP4. These results suggest that Neuroncell-EX is feasible and effective in promoting functional recovery and neuroprotection in ischemic rats, potentially through immunomodulation, angiogenesis, and neurogenesis mechanisms.