To investigate the effects of psychological stress on the masticatory muscles of rats, a communication box was applied to induce the psychological stress (PS) in rats. The successful establishment of psychological stimulation was confirmed by elevated serum levels of adrenocorticotropic hormone (ACTH) and changed behaviors in the elevated plusmaze apparatus. The energy metabolism of the bilateral masseter muscles was tested via chemocolorimetric analysis, whereas muscle ultrastructure was assessed by electron microscopy. In comparison to the control group, the PS group showed evidence of swollen mitochondria with cristae loss and reduced matrix density in the masticatory muscles after three weeks of stimulation; after five weeks of stimulation, severe vacuolar changes to the mitochondria were observed. Increased vascular permeability of the masticatory muscle capillaries was found in the five-week PS rats. In addition, there was decreased activity of Na(+)-K(+)ATPase and Ca(2+)-ATPase and a simultaneous increase in the activity of lactate dehydrogenase and lactic acid in the masticatory muscles of PS rats. Together, these results indicate that psychological stress induces alterations in the ultrastructure and energy metabolism of masticatory muscles in rats.