5-Fluorouracil (5-FU) is one of the frequently used chemotherapeutic agents against colorectal cancer (CRC). However, 5-FU treatment remains clinical challenges since a large fraction of patients with CRC developed resistance to 5-FU-based chemotherapies. Hexokinase 2 (HK II), coding for a rate-limiting enzyme of glutamine metabolism, is responsible for the dysregulated glycolysis of cancers. In this study, we report epidermal growth factor receptor (EGFR) and HK II were overexpressed in colon cancers and positively correlated with 5-FU resistance of CRC. In addition, expression of miR-143 was remarkedly suppressed in 5-FU resistant CRC patients and colon cancer cells. Moreover, miR-143 expression was effectively downregulated by EGFR and inversely associated with HK II expression in CRC cells. We identified HK II as a direct target of miR-143 in colon cancer cells. Overexpression of miR-143 inhibited glycolysis rate through direct targeting HK II, leading to re-sensitization of 5-FU resistant colon cancer cells to 5-FU treatment. Rescue experiments validated that recovering HK II in miR-143-overexpressing cells restored 5-FU resistance of CRC cells. In general, our study reveals critical roles of miR-143, which is a downstream effector of EGFR in 5-FU resistant CRC cells through direct targeting HK II, indicating miR-143 is an effectively therapeutic target for the treatment of patients with chemoresistant CRC.