Herein, we describe the establishment and characterization of the first mixed-phenotype acute leukemia cell line (JIH-5). The JIH-5 cell line was established from leukemia cells with B lymphoid/myeloid phenotype from a female mixed-phenotype acute leukemia patient. JIH-5 cells exhibit an immunophenotype comprised of myeloid and B lymphoid antigens. Whole-exome sequencing revealed somatic mutations in nine genes in JIH-5 cells. Transcriptional sequencing of JIH-5 cells identified EP300-ZNF384 fusion transcript, which is a recurrent alteration in B cell acute lymphoblastic leukemia. Our results suggest that the JIH-5 cell line may serve as a tool for the study of mixed-phenotype acute leukemia or EP300-ZNF384.