Summary Enhancement of antigen-specific T cell immunity has shown significant therapeutic benefit in infectious diseases and cancer. Hematopoietic progenitor kinase-1 (HPK1) is a negative-feedback regulator of T cell receptor signaling, which dampens T cell proliferation and effector function. A recent report showed that a catalytic dead mutant of HPK1 phenocopies augmented T cell responses observed in HPK1-knockout mice, indicating that kinase activity is critical for function. We evaluated active and inactive mutants and determined crystal structures of HPK1 kinase domain (HPK1-KD) in apo and ligand bound forms. In all structures HPK1-KD displays a rare domain-swapped dimer, in which the activation segment comprises a well-conserved dimer interface. Biophysical measurements show formation of dimer in solution. The activation segment adopts an α-helical structure which exhibits distinct orientations in active and inactive states. This face-to-face configuration suggests that the domain-swapped dimer may possess alternative selectivity for certain substrates of HPK1 under relevant cellular context. Highlights • The active and inactive HPK1 kinase in apo and ligand bound forms are characterized • Structures show rare domain-swapped dimer involving helical activation segment • Dimerization observed in solution suggesting potential biologically relevance • Ligand complex structure provides insights for structure-based drug design Enhancement of antigen-specific T cell immunity has shown significant therapeutic benefit in infectious diseases and cancer. Hematopoietic progenitor kinase-1 (HPK1) is one of the intracellular regulators that dampens T cell receptor signaling. Wu et al. studied the molecular structure of HPK1 kinase with bound ligand, which provides insights for structure-based drug design. [ABSTRACT FROM AUTHOR]