Hydroxyapatite/collagen (HAp/Col) nanocomposites with bone-like self-organized nanostructure show excellent bioactivity in vivo. However, they show quite high absorbability for cationic ions and lower culture medium ionic concentrations which adversely affects bone cell proliferation and osteogenic differentiation in in vitro cell culture condition. To address this limitation, in this study we have supplemented Ca2+ and Mg2+ ions to the HAp/Col nanocomposite membrane sample prior to cell culture to improve it’s in vitro biological properties. The HAp/Col nanocomposite membrane samples were fabricated by the simultaneous titration method using Ca(OH)2, type-I atelocollagen and H3PO4 as starting precursor materials. Prior to in vitro cell culture experiments, the HAp/Col samples were pretreated with Ca2+ and/or Mg2+ ions by immersing in 10 ml of 20 mM CaCl2 solution, 20 mM MgCl2 solution, or a solution containing 20 mM CaCl2 and 20 mM MgCl2 for 7 days. In vitro bone cell-material interactions on the pretreated and untreated HAp/Col samples were studied by culturing MC3T3-E1 cells up to 7 days. Enhanced bone cell proliferation was found on all the pretreated HAp/col samples as confirmed by the CCK-8 assay. Interestingly, the HAp/Col samples pretreated with both Ca2+ and Mg2+ ions showed the maximum viable bone cell density.