Mimicking the natural bone extracellular matrix, biomineralized nanofibers are envisioned as good choices for bone regeneration. Herein, composite nanofibers composed of poly(L-lactide) (PLLA) and gelatin (50/50, w/w) were electrospun and soaked in a modified five times simulated body fluid (SBF) for 6-24 h. Along with the soaking time, the amounts of deposited minerals increased, and the minerals transformed from dicalcium phosphate dehydrate (DCPD) to hydroxyapatite (HA). Mineral dissolution and Ca2+ ion release of these biomineralized nanofibers were investigated by putting them in deionized water or Hank's balanced salt solution. MC3T3-E1 osteoblasts were cultured in transwell chambers without contacting the materials or on the biomineralized nanofibers directly. In the noncontact culture, the released ions were found able to enhance osteogenic differentiation more significantly in comparison with cell proliferation. In the contact culture, all biomineralized nanofibers demonstrated strong ability in promoting both cell proliferation and osteogenic differentiation. Due to the fast dissolution of DCPD, the biomineralized nanofibers obtained from short SBF soaking was found to be inferior in enhancing osteogenic differentiation. Whereas the cells displayed high levels of alkaline phosphatase activity and collagen I synthesis when the material had abundant deposition of apatite. The results revealed that cell biological behaviors were synergistically influenced by the ionic dissolution products, the composition, and the morphology of biomineralized nanofibers, which could be regulated by timed biomineralization. (c) 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1968-1980, 2016.