Aberrant expression of histone deacetylases (HDACs) is associated with cancer drug resistance and tumor progression. While considerable studies and effort have been devoted to developing novel HDAC inhibitors in cancer therapy, hydroxamate-based HDAC inhibitors have gained growing interest for their broad-spectrum anti-tumor properties. We developed a series of HDAC inhibitors featuring a hydroxamate moiety, and WMJ-J-09 was selected due to its potent cytotoxic effect in colorectal cancer (CRC) cells, and its molecular mechanisms driving CRC cell death were characterized. WMJ-J-09 reduced cell viability, arrested the cell cycle at the G2/M phase, and triggered apoptosis. Mechanistically, it activated LKB1-p38MAPK signaling, leading to p53 phosphorylation and acetylation, which elevated p21 and suppressed survivin levels. WMJ-J-09 also acetylated α-tubulin, impaired microtubule assembly, and acetylated survivin, resulting in proteasomal degradation. Both LKB1 siRNA and anacardic acid, a histone acetyltransferase inhibitor, reversed WMJ-J-09-reduced survivin, confirming its dual effects on survivin at transcriptional and post-translational levels. In vivo, the subcutaneous growth of HCT116 CRC xenografts was reduced by WMJ-J-09. In conclusion, WMJ-J-09 causes CRC cell death via the LKB1-p53-survivin signaling pathway and HDAC inhibition, leading to acetylation of α-tubulin, p53, and survivin. This study highlights WMJ-J-09's potential as a promising therapeutic candidate for CRC treatment.