Osteosarcoma is most prevalently found primary malignant bone tumors, with primary metastatic patients accounting for approximately 25% of all osteosarcoma patients, yet their 5-year OS remains below 30%. Bilirubin plays a key role in oxidative stress-associated events, including malignancies, making the regulation of its serum levels a potential anti-tumor strategy. Herein, we investigated the association of osteosarcoma prognosis with serum levels of TBIL, IBIL and DBIL, and further explored the mechanisms by which bilirubin affects tumor invasion and migration.ROC curve was plotted to assess survival conditions based on the determined optimal cut-off values and the AUC. Then, Kaplan-Meier curves, along with Cox proportional hazards model, was applied for survival analysis. Inhibitory function of IBIL on the malignant properties of osteosarcoma cells was examined using the qRT-PCR, transwell assays, western blotting, and flow cytometry.We found that, versus osteosarcoma patients with pre-operative higher IBIL (>8.9 μmol/L), those with low IBIL (≤8.9 μmol/L) had shorter OS and PFS. As indicated by the Cox proportional hazards model, pre-operative IBIL functioned as an independent prognostic factor for OS and PFS in total and gender-stratified osteosarcoma patients (P < 0.05 for all). In vitro experiments further confirmed that IBIL inhibits PI3K/AKT phosphorylation and downregulates MMP-2 expression via reducing intracellular ROS, thereby decreasing the invasion of osteosarcoma cells.IBIL may serve as an independent prognostic predictor for osteosarcoma patients. IBIL impairs invasion of osteosarcoma cells through repressing the PI3K/AKT/MMP-2 pathway by suppressing intracellular ROS, thus inhibiting its metastatic potential.© 2023 The Author(s).