Oxidative stress (OS) has been implicated in a variety of pathological conditions, including diabetes mellitus, characterized by hyperglycemia. In the present study, OS induced by hyperglycemia and the effect of trolox, a vitamin E analog, were studied in cardiomyocytes and H9c2 cells exposed to 15 to 33 mM glucose (HG) for 24 to 72 hours in Dulbecco modified Eagle medium. Cells treated wirh 24 or 33 mM glucose for 24 hours or above showed decreased viability and adenosine triphosphate (ATP) content with a concomitant increase in radicals of oxygen species, calcium (Ca2+), mitochondrial permeability transition, and oxidative markers, confirming that the cells were under stress. However, upon exposure to 15 mM glucose for 24 hours, H9c2 cells maintained homeostasis and ATP generation. Pretreatment of cells with trolox reduced HG‐induced OS to control levels. Here, we report that the toxic effect of HG is highly regulated and that OS induction can be prevented with Trolox, a potential inhibitor of membrane damage.