In this study, a Ti–33Zr–12Al–6V alloy was prepared, and its work hardening and softening mechanisms were investigated via tensile tests conducted at room temperature. The interaction between the dislocations and strain-induced martensite (SIM) results in work hardening; whereas, the shearing of the grain boundaries by the dislocations for entering the adjacent grains results in work softening, which occurs when the logarithmic strains is between 5.8% and 7.6%. Work softening weakened the effect of work hardening; however, through work softening, the workability, which is insufficient in high-strength β-phase titanium alloys, was significantly increased. The microstructure evolution of Ti–33Zr–12Al–6V alloy under different strains was investigated using the quasi in-situ electron backscatter diffraction and scanning electron microscopy methods during the aforementioned tensile tests. The microstructure near the shear bands was observed via transmission electron microscopy using a focused ion beam. First, the {112}<111> slip systems with a high Schmid factor ( SF ) (＞0.25) were activated and, subsequently, the {112}<111> with a low SF (≤0.25) and {011}<111> slip systems were activated in most grains during tensile deformation. • The work hardening and softening behaviors were observed at the β-phase Ti–33Zr–12Al–6V high entropy alloy in ambient temperature tensile. • The quasi in-situ SEM and EBSD methods were applied to study the mechanism of work hardening and softening. • {112}<111 and {011}<111>slip systems were activated at different strain of tensile deformation.