The use safety of laser additive manufactured (LAM) titanium alloys is closely related to each one’s fracture failure mode. In this study, in situ tensile tests were carried out to study the deformation and fracture mechanisms of LAM Ti6Al4V titanium alloy before and after annealing treatment. The results showed that plastic deformation promoted slip bands to occur inside the α phase and shear bands to generate along the α/β interface. In the as-built sample, cracks initiated in the equiaxed grains and propagated along the columnar grain boundary, showing a mixed fracture mode. However, it transformed into a transgranular fracture after annealing treatment. The widmanstatten α phase acted as a barrier for slip movement and improved the crack resistance of grain boundaries.