Nanoparticles are widely used in various fields such as electronics, medicines and getting focus on the application in food industry for developing intelligent delivery system with bioactive ingredients or functional nutrients. Basic study on possible toxicological effect of food applicable nanoparticles is required for a practical application in food industry. In this study, size-controlled bovine serum albumin (BSA) nanoparticles were prepared by a desolvation method and their cytotoxicity was investigated. BSA nanoparticles were prepared with mean diameters as 115, 137, 159, and 299 nm, then cytotoxicity was evaluated with RAW 264.7 macrophages as in vitro model. Cell viabilities were significantly affected as increasing nanoparticle concentration. Smaller the sizes of nanoparticles, LD50 values were significantly reduced. LD50 values of BSA nanoparticles were 50, 65, 126, and 170 μg/ml, respectively. Nanoparticle was supposed to induce the apoptosis of RAW 264.7 marcrophages and underlying mechanism will be investigated in future. These findings will be used as valuable basement for nanofood development with BSA nanoparticles.