To evaluate the physiological potential of the defense system against hydroperoxidation of membrane-lipid components caused by environmental stresses in higher plants, we generated transgenic tobacco plants expressing a glutathione peroxidase (GPX)-like protein in the cytosol (TcGPX) or chloroplasts (TpGPX). The activities toward α-linolenic acid hydroperoxide in TcGPX and TpGPX plants were 47.5–75.3 and 32.7–42.1 nmol min−1 mg−1 protein, respectively, while no activity was detected in wild-type plants. The transgenic plants showed increased tolerance to oxidative stress caused by application of methylviologen (MV: 50 µm) under moderate light intensity (200 µE m−2 sec−1), chilling stress under high light intensity (4°C, 1000 µE m−2 sec−1), or salt stress (250 mm NaCl). Under these stresses, the lipid hydroperoxidation (the production of malondialdehyde (MDA)) of the leaves of TcGPX and TpGPX plants was clearly suppressed compared with that of wild-type plants. Furthermore, the capacity of the photosynthetic and antioxidative systems in the transgenic plants remained higher than those of wild-type plants under chilling or salt stress. These results clearly indicate that a high level of GPX-like protein in tobacco plants functions to remove unsaturated fatty acid hydroperoxides generated in cellular membranes under stress conditions, leading to the maintenance of membrane integrity and increased tolerance to oxidative stress caused by various stress conditions.