Purpose <p>Postoperative gut dysbiosis and inflammation are common complications in ovarian cancer patients. This study aims to investigate the protective role of probiotic therapy on gut microbiota homeostasis and its potential mechanism via activation of the ω-oxidation pathway.</p> Methods <p>We conducted a clinical analysis of ovarian cancer patients post-surgery who received probiotic therapy. Fecal samples were collected and analyzed using 16S rRNA sequencing to determine microbial diversity and composition. Metabolic pathway analysis was performed via MetaCyc. Additionally, an ovarian cancer mouse model was established to evaluate intestinal permeability, inflammatory cytokine levels, histopathological changes, and expression of ω-oxidation-related genes and proteins. The role of short-chain fatty acids (SCFAs) was also examined through combined administration with probiotics.</p> Results <p>Probiotic treatment significantly restored gut microbial diversity and reduced dysbiosis in post-surgical ovarian cancer patients. 16S rRNA sequencing revealed increased abundance of beneficial genera such as Alistipes, Roseburia, and Ruminococcus, while metabolic pathway analysis indicated significant activation of the ω-oxidation pathway. In mice, probiotic treatment reduced FD4 levels, restored intestinal barrier function, and modulated inflammatory cytokines by increasing IL-4 and IL-10 and decreasing IL-6 and TNF-α. Furthermore, probiotics upregulated expression of PPARα, CYP4F3, and CYP4A10 at both mRNA and protein levels, while reducing ROS and MDA levels, indicating alleviated oxidative stress. Combined treatment with SCFAs further enhanced these effects, suggesting a synergistic activation of ω-oxidation signaling and better restoration of gut homeostasis.</p> Conclusion <p>Probiotic therapy effectively restores gut microbiota composition and intestinal integrity following ovarian cancer surgery, potentially through activation of the ω-oxidation pathway.</p>