Human Y-Box Binding Protein-1 (YB-1) is overexpressed in breast cancer and regulates multiple oncogenic pathways involved in tumor progression and therapeutic resistance. We recently reported AIGM-2024–4 as a novel structure guided small molecule modulator of full length YB-1 protein. Here, we evaluated its preliminary in vivo tolerability in healthy female rats and antitumor activity in the DMBA-induced mammary tumor model. Acute oral toxicity assessment demonstrated that AIGM-2024–4 was well tolerated at doses up to 2000 mg/kg, with no detectable treatment related adverse effects under the tested conditions. In the DMBA-induced model, repeated oral administration of AIGM-2024–4 significantly reduced tumor growth, resulting in an ∼56% reduction in tumor volume compared with the disease control group. Treatment also reduced the mitotic index, improved redox balance, enhanced antioxidant status, and improved histopathological features of mammary tumors. These antitumor effects were accompanied by altered expression of YB-1 and tumor progression associated proteins, including HER2, EGFR, ERα, and Ki-67, together with increased E-cadherin expression. Collectively, this study provides an initial integrated in vivo evaluation of AIGM-2024–4, demonstrating favorable acute oral tolerability, preliminary antitumor activity, and YB-1- associated molecular changes that support further preclinical investigation.