Analgesic tolerance is partially mediated by enhanced glutamatergic transmission in the CNS. β-lactam antibiotics, through glutamate transporter subtype 1 (GLT-1) activation, reduce extracellular glutamate levels and attenuate tolerance to morphine analgesia in rats. Similar to opioids, nicotine has potent analgesic properties that are subject to tolerance. The purpose of this study was to evaluate the effects of ceftriaxone, a β-lactam antibiotic and GLT-1 activator on nicotine antinociception and its tolerance. Rats were pretreated for 5 days with ceftriaxone (200 mg/kg, intraperitoneally) before evaluating their analgesic response to nicotine (1.0 or 2.5 mg/kg, subcutaneously) for seven consecutive days using the tail-flick assay. Ceftriaxone-treated rats displayed an enhanced antinociceptive response to nicotine and unlike saline-injected controls, did not develop tolerance to nicotine's analgesic effects. These results suggest that GLT-1 transporter activation enhances and preserves nicotine antinociception and identify β-lactam antibiotics as potential complementary therapeutic agents for the treatment of chronic pain.