The effect of endogenous oxidative stress induced by γ-glutamyl cysteinesynthetase inhibitor D,L-buthionine sulfoximine (BSO) on the functioning of hypoxia-induced factor 1α (HIF-1α) was studied on Caco-2 cells. BSO was added for 24 h in concentrations of 5, 10, 50, 100, and 500 μM. It was shown that BSO in concentrations of 10, 50, and 100 μM induced endogenous oxidative stress and increased the content of HIF-1α; this effect was regulated through nuclear factor of erythroid origin 2 (Nrf2). Activation of HIF-1α had an independent protective effect, as evidenced by the decrease in cell viability after HIF-1α inhibition under these conditions. When the concentration of BSO was increased to 500 μM the content of HIF-1α did not change, and cell viability decreased.