Sepsis remains an important cause of mortality worldwide, and early deaths resulting from overwhelming inflammation in septic patients have been reported. Vigorous immune reactions are beneficial for bacterial clearance in this circumstance but at the price of self-tissue damage. Mesenchymal stem cells (MSCs) have been found to modulate immune function and attenuate sepsis. As the Toll-like receptor 4 pathway plays an important role in response to infections, here we investigated the mechanisms of MSC-mediated immunomodulation by determining the expression of Toll-like receptor 4 signaling in the liver and by circulating cytokines at 0, 1, 2, 3, and 6 h after cecal ligation and puncture (CLP)-induced sepsis in mice. We found that administration of umbilical cord-derived MSCs (UCMSCs) was beneficial for survival. Six hours after CLP, UCMSC administration decreased the expression of MyD88 mRNA and protein in the liver tissues of the mice, and also the ratio of NFκB phosphorylation (P = 0.041 and 0.005, respectively). Serum levels of TNF-α, MCP-1, IFN-γ, and IL-6 were significantly lower and IL-10 significantly higher 6 h after CLP in the mice receiving UCMSCs compared with those receiving PBS only. Our study provides the first in vivo evidence for the association of the MyD88-NFκB pathway and MSC-mediated immunomodulation during sepsis. The immunomodulatory effect of UCMSCs was noted from 3 to 6 h after injection, and the MyD88-NFκB pathway played an important role in response to the immunomodulatory signals from UCMSCs.