Numerous chemotherapeutic agents are currently employed in cancer treatment, but many are associated with significant side effects. This study aims to identify a novel anticancer drug that minimizes or eliminates these adverse effects. The anticancer activity of the Rhodium (III) complex cis-[RhLI2]I was evaluated through both in vivo and in vitro functional assays. Apoptosis in cancer cells post-treatment was assessed using microscopy and gene expression analysis. In cytotoxicity screening via the brine shrimp lethality bioassay, the compound exhibited an LC50 value of 25.90 µg/mL (P < 0.001). It also achieved an 88.96% inhibition of cell growth (P < 0.001), an 82.39% increase in lifespan (P < 0.001), and a significant reduction in tumor weight at a dosage of 200 µg/kg in Ehrlich ascites carcinoma (EAC)-bearing Swiss albino mice. Restoration of hematological parameters, such as RBC, WBC, and hemoglobin levels, was observed in treated tumor-bearing mice compared to untreated EAC-bearing mice. The compound inhibited the growth and proliferation of breast cancer (MCF7) cells in a dose-dependent manner, achieving a maximum inhibition of 88.9% at 200 µg/mL. Apoptotic induction in MCF7 cells occurred through the upregulation of p53, Bax, caspase-3, -8, and -9, alongside the downregulation of the anti-apoptotic protein Bcl-2. No long-term adverse effects on hematological or biochemical parameters or tissue levels were observed in the mice. Given these findings, this compound demonstrates significant cytotoxic effects and has the potential to serve as a promising chemotherapeutic agent, warranting further investigation at more advanced stages.