The purpose of this study was to evaluate whether berberine (BER) administration could attenuate depression- and anxiety-like behaviors and increase corticotrophin-releasing factor (CRF) and tyrosine hydroxylase (TH) expression following chronic morphine withdrawal in rats. Male rats were exposed to chronic, intermittent, escalating morphine (10∼50 mg/kg) for 10 days. After the last morphine injection, depression- and anxiety-like beahvior associated with morphine discontinuation persisted for at least three days during withdrawal without any change in ambulatory activity. Daily BER administration significantly decreased immobility in the forced swimming test and increased open-arm exploration in the elevated plus maze test. BER administration also significantly blocked the increase in hypothalamic CRF expression and TH expression in the locus coeruleus (LC) and the decrease in hippocampal brain-derived neurotrophic factor (BDNF) mRNA expression. Taken together, these findings demonstrated that BER administration significantly reduced morphine withdrawal-associated behaviors following discontinuation of repeated morphine administration in rats, possibly through modulation of hypothalamic CRF and the central noradrenergic system. BER may be a useful agent for treating or alleviating complex withdrawal symptoms and preventing morphine use relapses.