In metal additive manufacturing (AM) processes, due to the high cost of metal powder, it is common to reuse the collected powder from the build envelope for future builds. Powder reuse may adversely affect the powder characteristics, including the flowability, size distribution, chemical composition, resultant microstructural, and consequently, mechanical properties of the fabricated parts. This study aims to investigate the effect of powder reuse on the microstructural features and fatigue performance of Ti-6Al-4V specimens fabricated using a directed energy deposition (DED) process. Characteristics of reused powder particles, such as the size distribution and chemical composition, were evaluated and compared with that of virgin powder. Microstructural features and characteristics of the process-induced defects were examined using scanning electron microscopy and x-ray computed tomography, respectively. Fatigue performance of the specimens fabricated using reused powder was evaluated and compared to their control counterparts, fabricated using virgin powder.