Biodegradable amino acid ester-substituted polyphosphazenes are unique biomaterials for tissue engineering. Considering the surface properties as topography and chemical composition having vital roles in regulating cellular response, in this study, a kind of micropatterned polyphosphazene films were prepared and subjected to osteoblasts culture. Briefly, poly(glycine ethyl ester-co-alanine ethyl ester)phosphazene (PGAP) was synthesized, and its solution in chloroform was cast under high (80%) or low (20%) environmental humidity. Honeycomb-patterned or flat PGAP films were resulted. By analyzing with scanning electron microscope, atomic force microscope, X-ray photoelectron spectroscope, and water contact angle measurement, the honeycomb-patterned PGAP films demonstrated higher surface roughness, phosphorous and nitrogen content, and hydrophilicity than the flat one. Although the initial cell attachment and proliferation on PGAP films were inferior to those on conventional poly(lactic-co-glycolic acid) films, P-containing PGAP was a sort of bone-binding bioactive polymer. With these alternations, honeycomb-patterned PGAP films had accordingly enhanced protein adsorption and apatite deposition in simulated body fluid and showed great advantages in promoting osteogenous differentiation. The results suggested a potential way to make polyphosphazenes as good choices for bone tissue regeneration by increasing their surface roughness and phosphorous content. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.