To pursue a systemic administration of alpha-linolenic acid (ALA), which is a selective cytotoxic agent, we formulated an ALA o/w-emulsion stabilized by cholesterol-bearing pullulan (CHP-55-2.1) and trioctanoylglyceride (TriC8). This emulsion was stable even in the presence of bovine serum albumin (BSA). Peroxidation of ALA was drastically depressed by the emulsification using CHP. In addition, cytotoxic effect of the CHP/ALA/TriC8-emulsion against human colon cancer cell (RPM14788) was much higher than that of free ALA. However, no significant difference was observed in cell internalization efficiency of ALA between the two. These results suggest that difference in the cytotoxicity between the CHP/ALA/TriC8-emulsion and free ALA may come from difference in the intracellular behavior of ALA.