JAK/STAT pathway has been well confirmed in the development of colorectal cancer (CRC), however, the exact mechanism is unclear. Therefore, we aimed to identify key genes involved in JAK/STAT pathway in CRC, as well as the potential mechanism. RT² profiler PCR arrays were performed to identify key genes of the JAK/STAT pathway. GO, KEGG pathway and PPI analyses were performed to screen the main functions of differentially expressed genes (DEGs). Moreover, the expression of DEGs was detected by GEPIA based on TCGA database and verified by qPCR and/or Western blot. Subsequently, the association between the two DEGs (CXCL9 and IL6ST) and clinicopathological features were determined by immunohistochemistry, and survival analysis was also conducted. Finally, the effects of IL6ST overexpression on STAT3 activation and HT29 cell functions were analyzed. A total of 14 DEGs were identified. Among the DEGs, GHR, NR3C1, IL6ST and A2M were confirmed to be statistically decreased, while CXCL9 was significantly increased in the CRC tissues. Furthermore, CXCL9 was significantly associated with differentiation, lymph node metastasis, distant metastasis and invasion, while IL6ST was related with tumor size, differentiation, stage and invasion. Patients with high expression of IL6ST presented significantly lower lifetime, however, CXCL9 showed the opposite results without significance. Additionally, we found that overexpression of IL6ST statistically elevated p-STAT3 level, cell viability, adhesion rate and migration, and decreased apoptosis, but had no effects on cell cycle. Our results suggest that IL6ST is a critical key gene involved in JAK/STAT signaling pathway in CRC.Copyright © 2020 Elsevier Ltd. All rights reserved.