The control of IL-10 production in Toll-like receptor (TLR) signals remains to be elucidated. Here, we report that β-arrestin 2 positively regulates TLR-triggered IL-10 production in a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism. In vitro studies with cells including peritoneal macrophages and HEK293/TLR4 cells have demonstrated that β-arrestin 2 forms complexes with p38 and facilitates p38 activation after lipopolysaccharide (LPS) stimulation. Deficiency of β-arrestin 2 and inhibition of p38 MAPK activity both ameliorate TLR4-stimulated IL-10 response. Additionally, in vivo experiments show that mice lacking β-arrestin 2 produce less amount of IL-10, and are more susceptible to LPS-induced septic shock which is further enhanced by blocking IL-10 signal. These results reveal a novel mechanism by which β-arrestin 2 negatively regulates TLR4-mediated inflammatory reactions.