Osteosarcoma (OS) is an aggressive malignancy characterised by high metastatic potential and poor prognosis. Imipramine, a tricyclic antidepressant, has shown potential anticancer effects. This study evaluates the cytotoxic, pro-apoptotic and anti-invasion effects of imipramine on OS cells in vitro and in vivo, as well as its underlying mechanisms. Imipramine significantly reduced U-2 OS and MG 63 cell viability in a time- and dose-dependent manner, confirmed through MTT and colony formation assays. It induced apoptosis via caspase-dependent pathways, as evidenced by increased cleaved caspase-3, -8 and -9 levels and reduced expression of anti-apoptotic proteins such as MCL-1. Imipramine activated both extrinsic and intrinsic apoptosis pathways in vitro and in vivo, increasing Fas, Fas-L, BAX and BAK while suppressing anti-apoptotic factors like BCL-2 and XIAP. Transwell assays showed dose-dependent inhibition of cell migration and invasion, supported by suppressed Src phosphorylation and downregulation of EMT markers (Snail-1 and Slug). In U-2 OS xenograft-bearing mice, imipramine significantly inhibited tumour growth in a dose-dependent manner, with the 30 mg/kg group showing the smallest tumour volume and slowest growth rate (p = 0.0098). Tumour weights were significantly reduced without impacting body weight or liver and kidney function markers (AST, ALT, γ-GT and CREA). Histopathological analyses revealed no significant abnormalities in vital organs. Imipramine exerts potent anti-OS effects by suppressing Src-mediated invasion and enhancing caspase-dependent apoptosis through extrinsic and intrinsic pathways. It inhibits tumour progression without inducing systemic toxicity, demonstrating its potential as a therapeutic candidate for OS.