This present study investigated the effect of ultraviolet (UV) irradiation on bioactivity of micro-arc oxidized (MAO) titanium surface in vitro by cell culture medium immersion test and interactions with rat-derived mesenchymal stem cells (MSCs). UV-irradiated MAO surface exhibited no obvious changes in surface roughness, morphology, and phase composition when compared with MAO-only surface. However, in cell culture medium immersion test, markedly more bone-like apatite was formed on UV-modified samples than on MAO sample. Rat bone marrow- and adipose tissue-derived MSCs cultured on UV-modified samples displayed accelerated attachment, significant higher levels of alkaline phosphatase (ALP) activity, and up-regulated osteogenesis-related mRNA expression than MAO sample. XPS results provided direct evidence that the amount of basic hydroxyl groups increased with UV irradiation time, which could be one of the key mechanisms underlying their improved bioactivity.