Abstract: In this paper, the effect of building direction relative to the substrate (0°, 45°, and 90°) on the surface roughness of Ti6Al4V samples produced by selective laser melting (SLM) has been investigated. After SLM, the samples were subjected to different surface modifications, including acid etching (E) with HF, sandblasting (S) with Al2O3 and sandblasting + etching (SE). Surface roughness of the prepared samples were investigated by Alicona Infinite Focus optical microscopy and Scanning Electron Microscopy (SEM). It was found that there is an inverse relationship between surface roughness and building directions. Additionally, the results showed that the acid etching process increases the surface roughness of the samples at 45° and 90°, while the surface roughness decreases at 0°. Furthermore, the results showed that sandblasting and sandblasting followed with acid etching (SE) effectively reduced the surface roughness of the samples at all building directions. In summary, the acid-etched Ti6Al4V surfaces manufactured at 90° showed a superior surface roughness (Ra = 4.25 ± 0.21, Rz = 29.63 ± 2.34 μm) among the specimens, which is encouraged osseointegration of implants.