<p>Morphine, a potent opioid analgesic, is limited in clinical use due to tolerance, dependence, and withdrawal. Recent research highlights the gut microbiota as a crucial regulator of opioid effects through the microbiota-gut-brain axis. This study investigates the impact of antibiotic-induced suppression of the gut microbiota on morphine’s analgesic efficacy, tolerance, withdrawal, and related behavioral and neurophysiological changes in male mice. Mice received an 11-day antibiotic regimen followed by acute or repeated morphine administration. Behavioral assays (hot plate, tail flick, naloxone-induced withdrawal) evaluated analgesia, tolerance, and withdrawal, while anxiety-like behaviors were assessed via the elevated plus maze. Fecal samples were analyzed for colony-forming units, and EEG recordings measured brain activity. Antibiotics alone induced analgesia and prevented morphine tolerance in repeated dosing (<i>P</i> &lt; 0.001). Antibiotics also reduced withdrawal severity in chronic morphine-treated mice (<i>P</i> &lt; 0.05) but had no effect after acute morphine administration. Anxiety-like behaviors increased with antibiotics (<i>P</i> &lt; 0.001), and both acute (<i>P</i> &lt; 0.01) and chronic morphine (<i>P</i> &lt; 0.05), without interaction. Antibiotic treatment reduced fecal colony-forming units, most significantly with repeated morphine (<i>P</i> &lt; 0.001). Repeated morphine enhanced brain excitability, which was reduced by antibiotic pre-treatment. These findings suggest that modulating the gut microbiota may provide new strategies for managing opioid dependence and related disorders.</p>