Silicon-substituted hydroxyapatite (Si-HA) coatings have been plasma sprayed over titanium substrates (Ti-6Al-4V) aiming to improve the bioactivity of the constructs for bone tissue repair/regeneration. X-ray diffraction analysis of the coatings has shown that, previous to the thermal deposition, no secondary phases were formed due to the incorporation of 0.8 wt % Si into HA crystal lattice. Partial decomposition of hydroxyapatite, which lead to the formation of the more soluble phases of alpha- and beta-tricalcium phosphate and calcium oxide, and increase of amorphization level only occurred following plasma spraying. Human bone marrow-derived osteoblastic cells were used to assess the in vitro biocompatibility of the constructs. Cells attached and grew well on the Si-HA coatings, putting in evidence an increased metabolic activity and alkaline phosphatase expression comparing to control, i.e., titanium substrates plasma sprayed with hydroxyapatite. Further, a trend for increased differentiation was also verified by the upregulation of osteogenesis-related genes, as well as by the augmented deposition of globular mineral deposits within established cell layers. Based on the present findings, plasma spraying of Si-HA coatings over titanium substrates demonstrates improved biological properties regarding cell proliferation and differentiation, comparing to HA coatings. This suggests that incorporation of Si into the HA lattice could enhance the biological behavior of the plasma-sprayed coating.