An assessment is made of the anti-proliferative activity of cicada slough-derived materials against 10 human cancer cell lines, including PC-3 and DU145 prostate cancer cell lines, using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Results were compared with those of the commercially available anticancer agent with broad spectrum cisplatin. The ethanol extract of Cryptotympana spp. slough was proved to have anti-proliferative activity against A549 lung, AGS stomach, PC-3 and DU145 prostate, Hela cervix, HT-29 colon, MCF-7 breast, and SK-Hep-1 liver cancer cell lines except for Hep-2 larynx and SK-OV-3 ovary cancer cell lines. The biologically active constituent was characterized as the nonprotein α-amino acid theanine [2-amino-4-(ethylcarbamoyl)butyric acid] by spectroscopic analysis, including EI-MS and NMR. Theanine was isolated from the cicada slough as a new cytotoxic principle. Fifty percent inhibition concentration (IC50) values of the constituent against PC-3 was 6.52 μg/mL, respectively. The activity of theanine (IC50,6.52μg/mL) did not differ significantly from that of the anticancer agent cisplatin (IC50,7.39μg/mL) toward PC-3. In conclusion, further studies on the cicada slough-derived materials containing theanine as potential anticancer products or a lead molecule for the prevention or eradication from human prostate cancer.