Opioids remain the most effective analgesics despite their potential adverse effects such as tolerance and addiction. Mechanisms underlying these opiate-mediated processes remain the subject of much debate. Here we describe opioid-induced behaviors of Lmx1b conditional knockout mice (Lmx1bf/f/p), which lack central serotonergic neurons, and we report that opioid analgesia is differentially dependent on the central serotonergic system. Analgesia induced by a kappa opioid receptor agonist administered at the supraspinal level was abolished in Lmx1bf/f/p mice compared with their wild-type littermates. Furthermore, compared with their wild-type littermates Lmx1bf/f/p mice exhibited significantly reduced analgesic effects of mu and delta opioid receptor agonists at both spinal and supraspinal sites. In contrast to the attenuation in opioid analgesia, Lmx1bf/f/p mice developed tolerance to morphine analgesia and displayed normal morphine reward behavior as measured by conditioned place preference. Our results provide genetic evidence supporting the view that the central serotonergic system is a key component of supraspinal pain modulatory circuitry mediating opioid analgesia. Furthermore, our data suggest that the mechanisms of morphine tolerance and morphine reward are independent of the central serotonergic system.