: Colorectal cancer (CRC) remains a leading cause of cancer-related mortality globally, highlighting the urgent need for novel therapeutic strategies. This study aimed to investigate the anticancer potential of sodium pentaborate pentahydrate (NaB) in CRC by evaluating its effects on human colorectal cancer cell lines and elucidating underlying molecular mechanisms. : The cytotoxic and molecular effects of NaB were assessed in three human CRC cell lines (HCT-116, HT-29, and COLO-205) and one normal colon epithelial cell line (CCD-18CO). Cell viability assays were conducted to determine time- and dose-dependent responses. Apoptosis, cell cycle progression, colony formation, and migration capacity were evaluated. Gene and protein expression analyses were performed to examine apoptosis-related, DNA damage response, cell cycle, and Hippo signaling pathway components. : NaB significantly reduced cancer cell viability in a time- and dose-dependent manner, with minimal cytotoxicity to normal colon cells. It induced marked apoptosis, especially in HCT-116 and COLO-205 cells, and caused G2/M cell cycle arrest. In HCT-116 cells, NaB suppressed proliferation by downregulating PCNA and MKI-67 and reduced colony formation and migration. Molecular analyses revealed upregulation of pro-apoptotic BAX and downregulation of BCL-2, ATM, ATR, and cell cycle-related genes. NaB also inhibited oncogenic Hippo signaling by enhancing YAP1 phosphorylation and decreasing CTGF and CYR61 expression. : These findings demonstrate that sodium pentaborate pentahydrate exerts selective anticancer effects on colorectal cancer cells through the induction of apoptosis, cell cycle arrest, and suppression of key oncogenic pathways. NaB represents a promising candidate for further development as a therapeutic agent in CRC treatment.