In this study, using complementary in vitro and in vivo colon cancer models, we show for the first time that the MEK5/ERK5 pathway is an important modulator of 5-FU anticancer properties, the cornerstone agent in colon cancer treatment, further expanding the relevance of MEK5/ERK5 signaling in chemoresistance. Here, MAP2K5 is linked to malignant colon neoplasm.