The human umbilical cord is a promising source of mesenchymal stromal cells (MSCs). Intravenous administration of human umbilical cord-derived MSCs (IV-hUMSCs) showed a favorable effect in a rodent stroke model by a paracrine mechanism. However, its underlying therapeutic mechanisms must be determined for clinical application. We investigated the therapeutic effects and mechanisms of our good manufacturing practice (GMP)-manufactured hUMSCs using various cell doses and delivery time points in a rodent model of stroke. IV-hUMSCs at a dose of 1 × 10⁶ cells at 24 h after stroke improved functional deficits and reduced neuronal damage by attenuation of post-ischemic inflammation. Transcriptome and immunohistochemical analyses showed that interleukin-1 receptor antagonist (IL-1ra) was highly upregulated in ED-1-positive inflammatory cells in rats treated with IV-hUMSCs. Treatment with conditioned medium of hUMSCs increased the expression of IL-1ra in a macrophage cell line via activation of cAMP-response element-binding protein (CREB). These results strongly suggest that the attenuation of neuroinflammation mediated by endogenous IL-1ra is an important therapeutic mechanism of IV-hUMSCs for the treatment of stroke.