Atractylenolide I (ATL-I) can interfere with Colorectal cancer (CRC) cell proliferation by changing apoptosis, glucose metabolism and other behaviors, making it an effective drug for inhibiting CRC tumor growth. In this paper, we investigated the interactions between ATL-I and Keratin 7 (KRT7), a CRC-specific marker, to determine the potential pathways by which ATL-I inhibits CRC development. The KRT7 expression level in CRC was predicted online using the GEPIA website and then validated. HCT-116 cells' proliferation and invasion ability were determined using MTT and Trans well assays and the apoptosis rate using flow cytometry. Glucose consumption, lactate and ATP production levels were measured in HCT-116 cells using appropriate kits. Created the subcutaneous graft tumor model and identified the ATL-I effect in vivo. KRT7 was highly increased in CRC cells, KRT7 knockdown and overexpression prevented and encouraged malignant biological processes, as well as glycolysis. ATL-I acts by inhibiting the expression of KRT7, which can greatly inhibit the formation of CRC transplantation tumors in nude mice. ATL-I can impede the malignant biological process and glycolysis of CRC cells by inhibiting KRT7 expression in vitro and in vivo.