Intra-abdominal adhesion is a complication following abdominal surgery caused by the suppression of fibrinolytic activity and aggravated fibroblast invasion of the injured area, which may lead to chronic illnesses such as chronic pain, intestinal obstruction, and female infertility. This study hypothesized that lumbrokinase, a fibrinolytic enzyme extracted from the earthworm, supports the wound healing process. Therefore, we assessed the effect of lumbrokinase on intra-abdominal adhesion. Lumbrokinase treatment significantly decreased the severity and the area of intra-abdominal adhesion in a dose-dependent manner compared with the controls (untreated and hyaluronate-treated). Lumbrokinase-associated adverse effects were not observed. Immunohistochemical analysis of adhesion tissues revealed a loosened adhesive band between tissues, coupled with significantly decreased peritoneal thickening in the lumbrokinase-treated group versus the control group. Three-dimensional spheroid, MTT, and scratch wound migration assays using the IMR-90 human fibroblast cell line demonstrated that lumbrokinase significantly attenuated the migration and adhesive activity of fibroblasts without compromising cell proliferation. The luciferase assay and western blot analysis showed that lumbrokinase inhibited the AP-1/ICAM-1 cell adhesion signaling pathway. Therefore, lumbrokinase decreases intra-abdominal adhesion and peritoneal thickening by augmenting fibrinolytic action and inhibiting fibroblast migration and adhesive activity via attenuation of the AP-1/ICAM-1 signaling pathway. Lumbrokinase is thus a promising agent to prevent intra-abdominal adhesion.