Gamma-glutamyl transpeptidase (GGT) is an enzyme located on the surface of cellular membranes and involved in glutathione metabolism and maintenance of redox homeostasis. High GGT expression on tumor cells is associated with increased cell proliferation and resistance against chemotherapy. GGT inhibitors evaluated so far in clinical trials are too toxic for human use. In this study, using enzyme kinetics analyses, we demonstrate that ovothiols, 5(Nπ)-methyl thiohistidines of marine origin, act as non-competitive inhibitors of GGT with an apparent Ki of 21 μM, when we fixed the concentrations of the donor substrate. We found that these compounds are more potent than the known GGT inhibitor 6-diazo-5-oxo-L-norleucine and are not toxic toward human embryonic cells. In particular, cellular process-specific fluorescence-based assays revealed that ovothiols induce a mixed cell-death phenotype of apoptosis and autophagy in GGT-overexpressing cell lines, including human liver cancer and chronic B leukemic cells. The findings of our study provide the basis for further development of 5-thiohistidines as therapeutics for GGT-positive tumors and highlight that GGT inhibition is involved in autophagy.