The microstructural evolution and underlaying deformation behavior of Ti–6Al–4V alloy fabricated by laser powder bed fusion (LPBF‐Ti64) during room (RT) and cryogenic (CT: −150 °C) temperature are systematically investigated. LPBF‐Ti64 exhibits significantly higher strength at CT (yield strength [YS]: 1339 MPa, ultimate compressive strength [UCS]: 1982 MPa) as compared to RT (YS: 1013 MPa, UCS: 1690 MPa) with reduction in fracture strain. To probe the deformation mechanisms responsible for exceptionally higher strength at CT, LPBF‐Ti64 alloy is compressed to 10% strain at both temperatures and the underlaying strengthening mechanisms are investigated. Dislocation strengthening is emphasized to be the major strengthening mechanisms (1166/1359 MPa for RT‐10 and CT‐10, respectively). The significantly higher dislocation density at CT (3.73 1016 m−2 for CT‐10 and 2.9 1016 m−2 for RT‐10) is attributed to the restricted dislocation movement at low temperature, whereas dislocation nucleation during further deformation will multiply the dislocations interlocking and pinning, hence resulting in higher strength.