Our understanding of brain function under the drug treatment and pathology has been promoted by genetically encoded tools for labeling neuronal subpopulation and manipulating neuronal function. Recent studies have indicated that ventral tegmental area (VTA) dopamine (DA) neurons are heterogeneous in their properties. In the present study, we demonstrated that morphine (MRP)-responsive neurons were isolated from the VTA of c-Fos-EGFP/Rpl10a transgenic mice expressing an EGFP fused to Rpl10a, under the control of the c-fos promoter and purified using MACS and FACS. These experiences showed that systemic administration of MRP activated a subset of VTA neurons, including TH-positive DA neurons. Furthermore, using c-Fos-eNpHR mice, optical suppression of MRP-responsive VTA neurons significantly inhibited analgesic responses induced by systemic administration of MRP. These results suggest that the activation of MRP-responsive mesolimbic DA neurons partly modulates MRP-induced analgesia. The present findings provide evidence that labeling neuronal subpopulation and manipulating neuronal function using genetic engineering technology is very powerful tool for detecting functional neural circuits under drug treatment and disease conditions.