We report the cloning and characterization of a novel 54‐kDa high‐mobility group (HMG)‐box protein as the ligand for the human pan‐B cell co‐receptor CD19 (CD19‐L), which interacts with the extracellular domain of CD19 in trans. CD19‐L is the first CD19‐specific recombinant human protein with potent anti‐leukaemic activity against B‐lineage acute lymphoblastic leukaemia (ALL), the most common form of childhood cancer and the second most common form of acute leukaemia in adults. Soluble recombinant CD19‐L protein exhibited exquisite specificity for the extracellular domain of CD19 and strong binding to the surface of B‐lineage leukaemia/lymphoma cells. Engagement of CD19 co‐receptor on B‐lineage ALL cells with CD19‐L perturbed the CD19‐associated signalling network, altering the expression levels of multiple genes directly involved in regulation of apoptosis, and triggered rapid apoptotic cell death in a CD19‐specific manner. The identification of human CD19‐L may lead to therapeutic innovation for B‐lineage ALL and other B‐lineage lymphoid malignancies as well as B‐cell lymphoproliferative states and systemic autoimmunity.