Colorectal cancer (CRC), one of the diseases posing a threat to global health, according to the latest data, is the third most common cancer globally and the second leading cause of cancer-related deaths. The development and refinement of novel structures of small molecular compounds play a crucial role in tumor treatment and overcoming drug resistance. In this study, our objective was to screen and characterize novel compounds for overcoming drug resistance via the B Lymphoma Mo-MLV insertion region 1 (Bmi-1) reporter screen assay. The stable cell line harboring the Bmi-1 reporter gene was utilized to screen 300 compounds, leading to the identification of an amino-pyrimidine compound, APD-94. In vitro, APD-94 markedly inhibited cancer cell proliferation and decreased Bmi-1 expression at both the RNA and protein levels. In vivo, APD-94 repressed the growth of HT29 cell xenografts in NOD/SCID mice without notable side effects. Flow cytometry results demonstrated that APD-94 induced G2/M phase arrest and apoptosis in cells. APD-94 was identified as a novel inhibitor of microtubule polymerization by directly targeting the tubulin. Furthermore, APD-94 was more effective in overcoming the resistance to paclitaxel in paclitaxel-resistant A549/Tax cells. This bifunctional inhibitor is a promising candidate drug for CRC treatment.