Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal malignancies worldwide, with limited effective therapeutic options. Identifying novel targets is crucial for improving treatment strategies. In this study, we integrated bioinformatics and experimental approaches to uncover key regulatory genes in HCC. BIRC5 (survivin) emerged as a central hub gene, playing a pivotal role in apoptosis inhibition and cell cycle regulation. Using CRISPR-Cas9-mediated knockout in HepG2 cells, we demonstrated that BIRC5 depletion significantly suppressed cell proliferation and migration while inducing apoptosis. Furthermore, BIRC5 knockout led to cell cycle arrest, cytokinesis defects, and autophagy activation, highlighting its essential role in tumor maintenance. Functional assays, including colony formation, wound healing, flow cytometry, gene expression profiling, and transmission electron microscopy, validated these findings. Notably, the downregulation of key oncogenic pathways, including PI3K and AURKA, underscores the critical function of BIRC5 in sustaining HCC cell survival and proliferation. These results position BIRC5 as a promising therapeutic target, with the potential to disrupt tumor growth and metastasis. Targeting BIRC5 could offer a novel strategy for improving HCC treatment outcomes, paving the way for more effective therapeutic interventions against this aggressive cancer.