Chronic neuropathic pain poses a significant health problem worldwide, for which effective treatment is lacking. The current work aimed to investigate the potential analgesic effect of isoliquiritin, a flavonoid from Glycyrrhiza uralensis, against neuropathic pain and elucidate mechanisms. Male C57BL/6J mice were subjected to chronic constriction injury (CCI) by loose ligation of their sciatic nerves. Following CCI surgery, the neuropathic mice developed pain-like behaviors, as shown by thermal (heat) hyperalgesia in the Hargreaves test and tactile allodynia in the von Frey test. Repetitive treatment of CCI mice with isoliquiritin (p.o., twice per day for two weeks) ameliorated behavioral hyperalgesia to thermal (heat) stimuli and allodynia to tactile stimuli in a dose-dependent fashion (5, 15 and 45 mg/kg). The isoliquiritin-triggered analgesia seems serotonergically dependent, since its antihyperalgesic and antiallodynic actions were totally abolished by chemical depletion of spinal serotonin by p-chlorophenylalanine, whereas potentiated by 5-HTP (a precursor of 5-HT). Consistently, isoliquiritin-treated neuropathic mice showed escalated levels of spinal monoamines especially 5-HT, with depressed monoamine oxidase activity. Moreover, isoliquiritin-evoked antihyperalgesia and antiallodynia were preferentially counteracted by the 5-HT receptor antagonist WAY-100635 delivered systematically or spinally. Of notable benefit, isoliquiritin was able to correct co-morbid behavioral symptoms of depression and anxiety evoked by neuropathic pain. Collectively, these findings demonstrate, for the first time, the therapeutic efficacy of isoliquiritin on neuropathic hypersensitivity, and this effect is dependent on the spinal serotonergic system and 5-HT receptors.