trans-Anethole, the major bioactive component of Illicium verum Hook. commonly known as star anise exhibits various pharmacological activities including anti-inflammatory, antimicrobial, insecticidal, and antitumor. Osteosarcoma is an extremely aggressive malignant bone tumor that affects children and young adults and accounts for around 60% of all sarcomas. The study was planned to evaluate the potential of trans-Anethole against Human osteosarcoma cell line MG-63. The antiproliferative activity of trans-Anethole was assessed by MTT assay. trans-Anethole exhibited apoptotic cell death as monitored by confocal/electron microscopy and flow cytometry studies. Modulation of gene expression was studied by Western blot and RT-PCR analysis. The present study revealed that trans-Anethole inhibited osteosarcoma proliferation in a dose-dependent manner with a GI50 value of 60.25 µM and showed pro-apoptotic activity as analyzed by Annexin V-FITC/PI assay. Flow cytometric analysis revealed that trans-Anethole induced cell cycle arrest at the G0/G1 phase with the generation of reactive oxygen species and reduction in mitochondrial membrane potential (ΔΨm). Immunoblotting results showed the increased expression of caspase-9/-3, p53, and decreased expression of Bcl-xL suggesting the involvement of the p53 and mitochondrial intrinsic pathway. This work provides a rationale that trans-Anethole might be considered as a promising chemotherapeutic/nutraceutical agent for the management of osteosarcoma.Highlightstrans-Anethole inhibited cell growth and caused G0/G1 arrest in Human osteosarcoma MG-63 cell line.trans-Anethole led to the loss of mitochondrial membrane permeability along with ROS generation.trans-Anethole upregulates the expression of p53, Caspase-9/-3, and downregulate Bcl-xL expression.