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Vitamin E is a potent antioxidant that has been considered involved in fertility, but studies have mostly focused on α-tocopherol. Our study aimed at measuring, by an isotope dilution gas chromatography-mass spectrometry method, α- and γ-tocopherol concentration in human semen in a large and well-characterised population (134 men with different semen parameters and in varicocele patients), as well as their potential role in male fertility. We carried out freeze/thaw experiments in 15 samples with the two isomers in the cryoprotective medium. Moreover, our study included 10 subjects supplemented in vivo with α-tocopherol for 90 days. In seminal plasma, γ-tocopherol concentration was significantly lower in the varicocele group than in the normozoospermic group. We observed that γ-tocopherol, supplemented to cryopreservation medium, induced a higher post-thaw human sperm viability and motility than α-tocopherol. The results of in vivo α-tocopherol supplementation showed a decrease in γ-tocopherol concentration with increasing α-tocopherol level in blood. This is the first report related to γ-tocopherol distribution in human semen analysed by gas chromatography-mass spectrometry. γ-tocopherol would not seem to be related to semen parameters but to cellular oxidative condition. This tocopherol may contribute to human health in a yet unexplored way.