We analyzed the drug resistance of 5-Fu-resistant HCT8-R cells and demonstrated that the dietary flavonoid kaempferol enhanced the expression of miR-326 in colon cancer cells; we also showed that miR-326 could inhibit the process of glycolysis by directly targeting PKM2 3′-UTR to inhibit the expression of PKM2 or indirectly block the alternative splicing factors of PKM mRNA, and then reverse the resistance of colorectal cancer cells to 5-Fu. This evidence concerns the gene PKM and colorectal cancer.