The deformation behavior and microstructure characteristics of laser-welded Ti-6Al-4V titanium alloy joints under variable amplitude fatigue ranging from 320 to 380 MPa (initial maximum stress) were investigated in steps of 10 MPa. Each fatigue test included three different stages: the maximum stress amplitude was initial one for the first stage, but increased by 1.2 and 1.4 times for the second and third stages, respectively. The results showed that the variable amplitude fatigue limit of laser-welded the Ti-6Al-4V joint was 339 ± 5 MPa. Fractography analysis revealed that the fatigue cracks originated from the surface in the weld metal (WM) and were caused by the differences of grain size and texture orientation between WM and heat-affected zone. The average grain size of WM was significantly larger than that of other regions. Acicular α’ martensite was distributed mainly in the coarse grains of WM and its orientation was more concentrated, leading to the preferred orientation. Furthermore, the accumulation of dislocations on the acicular α’ and β phase interface led to fatigue crack initiation owing to stress concentration. In addition, the lager average Schmid factor and smaller deviation in WM of the joint at a large stress amplitude was owing to the smaller relative geometrical orientation. •The variable amplitude fatigue behavior of laser-welded Ti-6Al-4V joint was studied.•Differences in grain size and orientation lead to joint fracture in weld metal.•Dislocations accumulated on the phase interface are key elements leading fracture.•The characteristics of joint microstructure and fracture behavior are related.