We recently demonstrated an increase in spinal cerebrospinal fluid (CSF) excitatory amino acids (EAAs) in morphine-tolerant rats after morphine challenge. The present study examined whether co-infusion of the glucocorticoid dexamethasone (DEX) co-infusion inhibited morphine tolerance and the morphine challenge-induced EAAs increase after long-term morphine infusion. Intrathecal (i.t.) catheters and one microdialysis probe were implanted to male Wistar rats. Rats were divided into four groups: i.t. morphine (15μg/h), saline (1μl/h), DEX (2μg/h), or DEX (2μg/h) plus morphine (15μg/h) infusion for 5 days. Tail-flick responses were examined before drug infusion and daily after the start of infusion for 5 days. Moreover, on day 5 after morphine challenge (50μg, i.t.), CSF EAAs was also measured. Rat spinal cords were removed on day 5, and prepared for Western blot analysis of different glutamate transporters (GTs). The AD₅₀ (analgesic dose) on day 5 was 1.33μg in saline-infused rats, 83.84μg in morphine-tolerant rats, and 10.15μg in DEX plus morphine co-infused rats. Single DEX (2μg, i.t.) injection did not enhance morphine’s antinociceptive effect in either naïve or morphine-tolerant rats. No difference in CSF EAA level was observed in all groups between baseline (before drug infusion) and on day 5 after tolerance developed. Surprisingly, on day 5, after morphine challenge, an increase in glutamate and aspartate (284±47% and 201±18% of basal) concentration was observed, and morphine lost its antinociceptive effect (maximum percent effect, MPE=41±12%), whereas DEX/morphine co-infusion inhibited morphine-evoked EAA increase with a MPE=97±2%. DEX co-infusion prevented the downregulation of glial glutamate transporters (GLAST (Glu-Asp transporter) and GLT-1 (Glu transporter-1)), but not the neuronal GT EAAC1 (excitatory amino acid carrier). Upregulation of GLT-1 was also observed (204±20% of basal). DEX co-infusion inhibits the morphine-challenge induced EAA increase and prevents the loss of morphine’s antinociceptive effect after long-term morphine infusion.