Abstract: Antitumor effect of methylene blue in vitro were tested by methods of directkilling and inhibition of cell growth and clonogenic survival, Both dose and timedependent were the main character of It's antitumor efficiency, The surviving frac-tion of human lung adenocarcinoma cell line LTEP-a3 at concentration of 1 ug/ml of this dye treating for 4 hours was 10^-1,48 hours 10^-2,72 hours 10^-3,prolong-ing treating time at concentration of 1 ug/ml or more of methylene blue could inc-rease it's killing efficiency for cancer cells obviously, When the btreating timewere prolonged to 24 hours or more, the killing efficiency were related closelyto Its concentration, The damage location of cancer cells by meihylene blue wereobserved mainly in- mitochondria.