Janus kinase 2 (JAK2) is an important mediator of cytokine receptor signaling and plays key roles in hematopoietic and immune responses. The acquired JAK2 R683G(S) somatic mutations are detected in 15% of patients with B-cell acute lymphoblastic leukemia (B-ALL) and are presumed to be a biomarker for B-ALL. However, how JAK2 R683G(S) mutations lead to B-ALL is still unclear. Our results indicated that the E627 and R683 interaction played a vital role in JAK2 autoinhibition. Mutations (R683S, R683G and E627A) disrupting this interaction led to JAK2 constitutive activation, while mutations (R683K, E627D) restoring this interaction decreased its activity. Furthermore, spectroscopy experiments implied that disruption of the E627 and R683 interaction abolished JH1/JH2 domain interactions and forced the JH1 domain into the open, active conformation. Mutations abolishing this interaction promoted the proliferation of Ba/F3 cells. The results herein may provide clues to understanding the mechanism of JAK2 R683G(S) mutation-associated B-ALL.