Our study reveals the intricate mechanisms underlying the inhibition of invasive and metastatic capabilities in drug-resistant CRC cells by H. erinaceus erinacine S. This inhibition is achieved through the suppression of the macrophage migration inhibitory factor (MIF)/ C-X-C Motif Chemokine Receptor 4 (CXCR4)/ phosphoinositide 3-kinases (PI3K)/ protein kinase B (Akt)/ extracellular signal-regulated kinase (ERK)/ nuclear factor kappa B (NFκB)/HIF-1α) pathway and the reversal of EMT in HCT-116/5-fluorouracil-resistant (5FUR) cancer cells. Here, CXCR4 is linked to cancer.