We have used zinc aluminate nanostructured films deposited by spray pyrolysis to determine its biocompatibility assessed by cells attachment and cell differentiation. Cell attachment onto zinc aluminate showed an increase of 53, 81 and 86% at 180, 300 and 420 minutes (p<0.05) when compared to controls. Mineralization was analyzed at 5 and 14 days of culture by scanning electron microscopy, microanalysis and atomic force microscopy. Our results showed in experimental culture a higher density of mineral-like tissue with small needle-shaped crystal and granular nanoparticles with preferential orientation when compared to controls. The composition of the mineral-like tissue deposited in zinc aluminate nanostructured material had a Ca/P ratio of 1.6, whereas control culture had a Ca/P ratio of 1.50. Our finding revealed that ZnAl2O4 promoted higher expression of type I collagen, bone sialoprotein, osteocalcin and alkaline phosphatase, suggesting that zinc aluminate provides a microenvironment that favors mineral formation and cell differentiation. Our results point to the potential use of ZnAl2O4 for the osteoinductive process in biomedical implants.