This study aimed to investigate for the first time, the potential role of stevia aqueous extract alone and in combination with MK-801 on nalbuphine-induced tolerance and dependence in mice using biochemical and histological tools. In this study, Repeated administration of stevia extract (300 mg/kg, p.o) along with nalbuphine (10 mg/kg, s.c.) attenuated the development of tolerance, as measured by the hot plate test, and dependence, as assessed by naloxone (5 mg/kg, i.p.) - precipitated withdrawal manifestations. Concomitantly, increase in brain glutamate level, malondialdehyde level, and serum nitrite level-induced by repeated administration of nalbuphine to mice or by administration of naloxone to nalbuphine-dependent mice were inhibited by co-administration of stevia extract. Also, co-administration of the stevia extract inhibited the decrease in brain intracellular reduced glutathione level and glutathione peroxidase activity induced by both treatments. The inhibitory effect of the extract on nalbuphine-induced tolerance and dependence and on naloxone-induced biochemical alterations in nalbuphine-dependent mice was enhanced by concurrent i.p. administration of the NMDA receptor antagonist, MK-801 (0.25 mg/kg). histopathological results revealed that stevia extract co-administration produced decrease in nerve cell degeneration, vacuolation, apoptosis and enhance neuropil appearance. This histopathological improvement increased by concurrent administration of MK-801 with stevia extract. These results provide evidence that stevia extract appears to have a therapeutic potential in opioid tolerance and dependence, through inhibition of nalbuphine-induced elevation in glutamate level, NO overproduction and oxidative stress.