In vitro bone tissue development requires mimicking the in vivo bone environment, as well as a suitable combination of cells and scaffold for optimal results. We developed heterogeneous multilayered gelatin scaffolds with diverse compositions of porous structures by using a stacking procedure in which each layer had a different pore size depending on the gelatin concentration used. We then used these gelatin scaffolds to investigate the in vitro effect of varying porous structural compositions on the diffusion characteristics and cellular activity of MC3T3-E1 cells. We have shown that multilayered scaffolds with a larger pore size on the outer layers exhibited enhanced diffusion characteristics such as the diffusion coefficient compared to other scaffolds, including single-layered scaffolds with single pore size and multilayered scaffolds with a smaller pore size on the outer layers. Moreover, multilayered scaffolds with a larger pore size on the outer layers promoted cell adhesion, proliferation, and differentiation of MC3T3-E1 preosteoblast cells by providing a favorable environment for the cells within the tissue-engineered scaffold.