To broaden the knowledge of cytotoxicity of natural rubber latex (NRL) nanoparticles we for the first time examined the latex biocompatibility in vitro against mouse calvaria preosteoblastic cells (MC3T3-E1) and human alveolar basal epithelial (A549) cells. For NRL nanoparticles, the half maximal inhibitory concentration (IC50) value for MC3T3-E1 cells is one order of magnitude higher in toxicity as compared to that of A549 cells (3.99 mg/mL for MC3T3-E1 and 0.33 mg/mL for A549 cells). Owing to fractionation of NRL nanoparticles by ultra-centrifuge, the effect of the non-rubber constituents on the cytotoxicity was clarified. The suppression on the proliferation for A549 cells incubated with NRL nanoparticles was demonstrated by the cell cycle distribution. The in vitro study on osteogenic differentiation and expressions of proteins and characteristic genes of MC3T3-E1 cells demonstrated the promising results of the NRL nanoparticles for application in bone tissue engineering.