Bcr-Abl independent resistance to tyrosine kinase inhibitor (TKI) is a crucial factor lead to relapse or acute leukemia transformation in chronic myeloid leukemia (CML). However, its mechanism is still unclear. Herein, we found that of nine common protein kinases C (PKCs), PKC-β overexpression was significantly related with TKI resistance. Blockage of its expression in CD34+ cells and CML cell lines increased sensitivity to imatinib. Then, eighty-four leukemia related genes were compared between TKI-resistant CML cell lines with PKC-β silenced or not. Gene Ontology term and Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that Arachidonate 5-lipoxygenase (Alox5) and its relative pathway mainly participated in the resistance induced by PKC-β overexpression. It’s also observed that Alox5 was increased not only in bone marrow biopsy but also in CD34 cells derived from IM-resistant CML patients. The signaling pathway exploration indicated that ERK1/2 pathway mediates Alox5 upregulation by PKC-β. Meanwhile, we also proved that Alox5 induces TKI-insensitivity in CML through inactivation of PTEN. In vivo experiment, PKC-β elective inhibitor LY333531 prolonged survival time in CML-PDX mice model. In conclusion, targeted on PKC-β overexpression might be a novel therapy mechanism to overcome TKI-resistance in CML.