The intestinal barrier is crucial for homeostasis. This study aimed to investigate the protective effects of earthworm protein hydrolysates (EWPH) on the intestinal mucosal barrier and elucidate the underlying mechanisms. We first hydrolyzed earthworm protein using alcalase and identified the primary peptide components of EWPH through Nano LC-MS/MS analysis. Network pharmacology and bioinformatics approaches were employed to predict potential targets associated with the intestinal mucosal barrier. Experimentally, we demonstrated that EWPH effectively protects against dextran sulfate sodium (DSS)-induced intestinal barrier damage in mice. The protective mechanisms involve not only the inhibition of the Toll-like receptor 4 (TLR4)-nuclear factor-κ (NF-κ)/mitogen-activated protein kinases (MAPK) signaling pathway in the intestinal epithelium but also the suppression of other key molecules implicated in intestinal mucosal barrier damage, including phosphorylated-SRC proto-oncogene (p-SRC), phosphorylated-signal transducer and activator of transcription 3 (p-STAT3), Caspase-3, and matrix metalloproteinase-9 (MMP9), thereby mitigating intestinal inflammation and mucosal barrier injury. This study provides evidence that EWPH have the potential to safeguard the intestinal barrier hemostasis.