The effect of post-deposition heat treatment on microstructure and fatigue crack growth has been analyzed for electron beam melted Ti-6Al-4V plates. Samples have been heat-treated at temperatures of 950 °C and 1050 °C, and subsequently cooled at different cooling rates in the furnace and the water. The as-built sample possesses columnar prior β grains filled with exceptionally fine α+β Widmanstätten patterns and epitaxially grows in the vertical direction. Heat treatment with a slow cooling rate increases the α lath thickness, whereas fast cooling results in multiple needle-shaped α/α′ phases inside the prior β grains. The yield strength and ultimate tensile strength in as-built conditions are greater than the extruded mill annealed sample. The as-built samples show better crack growth resistance during the fatigue crack growth rate test than the heat-treated and mill-annealed samples. The lower plastic deformation of the as-built sample than the mill-annealed sample is attributed to the existence of fine α laths that restrict the motion of dislocation.