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.