<p>AHNAK nucleoprotein 2 (AHNAK2) is implicated in tumor progression and survival signaling, yet its role in chemotherapy resistance, particularly in colorectal cancer (CRC), remains under investigation. In the present study, the GEPIA database and Kaplan–Meier Plotter database were employed to uncover the correlation between high AHNAK2 expression and unfavorable prognostic outcomes in CRC patients. The expression of AHNAK2 in 5-fluorouracil (5-FU)-resistant CRC tissues was validated by immunohistochemical staining, quantitative real-time PCR, and western blot analysis. Then, 5-FU-resistant CRC cell lines LoVo/5-FU and HCT116/5-FU were developed through consecutive treatment of cells with 5-FU and then subjected to gene knockdown or overexpression. A series of assays, including CCK-8 assay, colony formation assay, flow cytometry, wound healing assay, transwell assay, and tumor xenograft mouse model, were conducted to evaluate the effects of AHNAK2 on 5-FU resistance. We observed a significantly increased expression of AHNAK2 in 5-FU-resistant tumor tissues compared to 5-FU-sensitive ones. This elevated expression was negatively associated with the prognosis of CRC patients. Knockdown of AHNAK2 in LoVo/5-FU cells reduced 5-FU resistance in CRC, whereas overexpression of AHNAK2 in HCT116/5-FU cells promoted resistance, both in vitro and in vivo. Mechanistically, AHNAK2 knockdown suppressed the expression of proteins such as PCNA, CDK4, p-AKT/AKT, and p-GSK-3β/GSK-3β, while enhancing the expression of cleaved caspase-3 and E-cadherin in LoVo/5-FU cells. Conversely, AHNAK2 overexpression in HCT116/5-FU cells produced the opposite effects. Collectively, these findings demonstrate that AHNAK2 reduces the chemosensitivity of CRC to 5-FU by activating the AKT/GSK-3β signaling pathway, underscoring its potential as a therapeutic target to improve CRC treatment strategies. </p>