This paper investigates the fatigue behaviour of laser powder bed fusion (LPBF) processed Ti6Al4V samples under three build orientations. The post-heat treatment (PHT-1050 °C) was carried out. The microstructural characterization was performed using optical microscopy, X-ray diffraction SEM and EDS techniques. The tensile test and high cycle fatigue tests were performed. The PHT performed at 1050 °C exhibited Widmanstatten microstructure consisting of a higher volume fraction of elongated β and a small amount of α. PHT samples’ ductility was ~ 67%, 40% and 177% higher than the as-printed samples under horizontal, inclined and vertical orientations. Interestingly, the fatigue lives of PHT samples at higher stress levels were higher and nearly isotropic in all three build orientations than the as-printed samples due to enhanced ductility and fewer critical pores. Further strong correlation between PHT samples and ductility was established. Moreover, there was a marginal improvement in fatigue limit due to PHT at 1050 °C compared to as-printed samples.