Colorectal cancer (CRC) ranks among the top three most prevalent malignancies globally and is a leading cause of cancer-related mortality. Traditional therapeutic approaches usually cause significant adverse effects, highlighting the urgent demand for alternative, more effective treatments. Probiotics have gained attentions as potential cancer therapy due to their beneficial impacts on host health. Clostridium butyricum (Cl. butyricum) has shown anticancer properties in recent studies, though the underlying mechanisms remain inadequately understood. This study presents an integrative analysis of network pharmacology and proteomics to elucidate the key targets of Cl. butyricum in CRC treatment. The network pharmacology analysis identified 72 overlapping genes, and functional analysis of these genes indicated that most pathways were related to pathways in cancer and inflammation, and butyrate emerging as the pivotal product of Cl. butyricum due to its strong associations with the identified hub genes. In parallel, proteomics analysis revealed 168 differential expressed proteins (DEPs) in Cl. butyricum-treated HCT-116 cells, comprising 78 upregulated and 90 downregulated proteins. These DEPs were primarily enriched in apoptosis and inflammatory pathways. PPI analysis further highlighted NFKB1 as key contributors to the anticancer effects of Cl. butyricum. The integrative analysis revealed a significant convergence of pathways enrichment patterns, particularly in inflammatory and immune-related pathways. Computational and experimental validation identified NFKB1 as a pivotal molecular target in CRC intervention. These collective findings elucidate the mechanistic basis of the antitumor properties of Cl. butyricum, highlighting its regulatory effects on NFKB1 through both inflammatory and, to a lesser extent, immunoregulatory pathways.