CDK8 is a transcriptional cyclin-dependent kinase and considered as a potential target in colon cancer therapeutics. Here, a novel selective CDK8 inhibitor was identified against colon cancer <i>in vivo</i>. Specifically, based on the structural information of the sorafenib-bound CDK8 structure, a series of novel 2-amino-pyridine derivatives were designed, synthesized, and evaluated. Among them, compound <b>29</b> showed strong inhibitory activity against CDK8 with an IC<sub>50</sub> value of 46 nM and favorable selectivity. And there is an apparent interaction between the endogenous or overexpressed CDK8 and biotinylated-<b>29</b>. This compound exhibited antiproliferation potency on colon cancer cell lines with a high CDK8 expression level, suppressed the activation of WNT/β-catenin and transcriptional activity of the TCF family, and induced G1 phase arrested in HCT-116 cells. In addition, this compound showed potent activity against sorafenib-resistant HCT-116 cells. What's more, it exhibited low toxicity and suitable pharmacokinetic (PK) profiles and showed preferable antitumor effects <i>in vivo.</i>