Biological performances such as osseointegration and biocompatibility of Ti6Al4V alloy primarily depends on topological and chemical properties of the surface of the bio-material. Here, a nanosecond pulsed Nd:YAG laser has been used to generate microstructures on Ti6Al4V surface by irradiating with 6000 number of laser shots per site. Formation of ripple structure and generation of sub-oxide phases on laser treated titanium surface supported uniform and dense growth of HAP on the sample. In contrast, discrete nucleation of HAP with comparable higher precipitation of calcium occurred on untreated Ti6Al4V sample when subjected to similar in vitro tests by exposing the sample to simulated body fluid. Initial interaction and growth of U2OS cells on untreated and laser treated Ti6Al4V substrates were quantified using MTT assay. More numbers of cell were attached to laser treated sample in comparison to untreated sample as observed in confocal microscope images. Our results suggested that surface patterning of Ti6Al4V alloy using nanosecond pulsed laser promoted bio-integration without compromising its biocompatibility. Copyright © VBRI Press.