The electron beam melting-printed Ti6Al4V shows a great potential application for orthopedic implants and aerospace in recent years. A systematic study on the microstructure of additive manufactured Ti6Al4V by electron beam melting both parallel to and perpendicular to the building directions (Z axis) is presented in the present investigation. The results showed that the microstructure of the alloy was α lamina with HCP structure and β bar with BCC structure. The original β phase grew as columnar crystal along the direction of construction, showing an equiaxial shape in the cross section, numerous small α lamellae block the original β phase, and presenting a cluster distribution on the original β grain boundary, and a basket-like distribution in the original β grain. This may be due to the rapid cooling of the small pool after melting, the repeated heating of the subsequent constructed layer on the formed layer, and the subsequent limited vacuum cooling, resulting in the formation of the micro morphology, which leads to the original β grain boundaries broken, and the formation of a distinctive basket or widmanstatten structure [1, 2]. In addition, XRD results indicated that there was α′ martensite, part of which has been decomposes into α phases and β phases, SEM and TEM experiments also proved this. Of note is that random distribution dislocation was observed in TEM. Using EBSD results, and it may be understand that the sample build direction was parallel to [0001] crystal orientation and build plane parallel to (1210) and (1100) crystal facets.