P38/Mk2 (mitogen-activated protein kinase (MAPK)-activated protein kinase-2, also known as MAKAP kinase-2) is a member of the mitogen-activated protein kinases (MAPKs) family, and participates in inflammatory responses directly or indirectly. WIN55, 212-2 (WIN55) is a synthetic non-selective agonist of cannabinoid (CB) receptors with remarkable anti-inflammatory properties. This study was to explore the roles of WIN55 and p38/Mk2 signaling pathway in dextran sodium sulfate (DSS)-induced mouse colitis and ascertain their anti-inflammatory mechanisms. Colitis was induced in C57BL Mk2 gene homozygous deletion (Mk2-/-) and wild-type mice by replacing the drinking water with 4% DSS solution for 7 days. DSS-treated mice developed bloody stool, weight loss, and eye-visible multiple bleeding ulcers on colon mucosa. The mRNA expressions levels of TNF-α and IL-6, as well as the protein levels of p38 and its phosphorylated form (p-p38), were upregulated in the colon. The plasma levels of TNF-α, IL-6, cytokine-induced neutrophil chemoattractant-1 (CINC-1), monocyte chemoattractant protein-1 (MCP-1), and lung myeloperoxidase (MPO) activities were raised; however, all these changes were less severe in Mk2-/- mice. After WIN55 intervention, the Mk2-/- mice recovered faster and better from the induced colitis than their wild-type counterparts. The results indicate that the Mk2 homozygous deletion in mice impedes the induction of experimental colitis by DSS, confirming the notion that p38/Mk2 is involved in this inflammatory response. WIN55 protects mice against DSS-induced colitis, in particular when the p38/Mk2 pathway is obstructed, implying that the activation of CB system, together with blocking of p38/Mk2 pathway, serves as a potential drug target for colitis treatment.