Abstract: Objective To investigate the effect of dasatinib (BMS-354825) on the proliferation of chronic lymphocytic leukemia (CLL) cell lines and the activation and function of wild-type/ibrutinib-resistant bruton tyrosine kinase (BTK) in vitro.Methods The inhibitory effect of dasatinib at different concentrations on the proliferation of CLL cell lines were observed. After transfection of indicated plasmids (wild-type BTK and its mutants BTKC481S, BTKT474F and BTKT474I/C481S) and treatment with dasatinib and ibrutinib, the phosphorylation levels of BTK and its initial downstream target PLCγ2 were measured by Western blot.Results The proliferation of CLL cell lines MEC-1 and JVM3 cells were significantly inhibited by dasatinib in a dose-dependent manner with an IC50 value of 3.54 mol/L and 0.65 mol/L, respectively. The phosphorylation of wild-type BTK and PLCγ2 was inhibited by 0.5 mol/L ibrutinib and 0.2 mol/L dasatinib. Upon activation of the BCR signaling, BTKC481S, BTKT474F and BTKT474I/C481S were functionally activated and phosphorylated its downstream target PLCγ2. All the mutations were resistant to ibrutinib at physical concentrations. Furthermore, 0.5 mol/L dasatinib inhibited the activation of BTKC481S followed by inhibition of PLCγ2 activation, but did not suppress the activation of BTKT474F and BTKT474I/C481S, which suggested that dasatinib might overcome BTKC481S-induced resistance to ibrutinib, without effect on "gatekeeper" BTK alterations.Conclusions Dasatinib inhibits the activation and function of both wild-type BTK and BTKC481S and hence inhibits the proliferation of CLL cells, which is promising to overcome ibrutinib resistance caused by BTKC481S.