The mu opioid receptor (MOR) modulates nociceptive pathways, reward processing, and mediates the strong analgesic and addictive properties of both medicinal as well as abused opioid drugs. MOR function has been extensively studied, and tools to manipulate or visualize the receptor protein are available. However, circuit mechanisms underlying MOR-mediated effects are less known, because genetic access to MOR-expressing neurons is lacking. Here we report the generation of a knock-in -Cre mouse line, which allows targeting and manipulating MOR opioid-responsive neurons. A cDNA encoding a T2A cleavable peptide and Cre-recombinase fused to enhanced green fluorescent protein (eGFP/Cre) was inserted downstream of the gene sequence. The resulting Cre line shows intact gene transcription. MOR and eGFP/Cre proteins are co-expressed in the same neurons, and localized in cytoplasmic and nuclear compartments, respectively. MOR signaling is unaltered, demonstrated by maintained DAMGO-induced G protein activation, MOR function is preserved as indicated by normal morphine-induced analgesia, hyperlocomotion and sensitization. The Cre-recombinase efficiently drives expression of Cre-dependent reporter genes, shown by local-virally-mediated expression in the medial habenula and brain wide fluorescence upon breeding with tdTomato reporter mice, the later showing a distribution patterns typical of MOR expression. Finally, we demonstrate that optogenetic activation of MOR neurons in the ventral tegmental area of -Cre mice evokes strong avoidance behavior, as anticipated from the literature. The -Cre line is therefore an excellent tool for both mapping and functional studies of MOR-positive neurons, and will be of broad interest for opioid, pain and addiction research. Here we develop an innovative tool to characterize circuit mechanisms underlying opioid actions, which may help the research communities to improve the knowledge on circuitry adaptation and response to opioid. The tool is particularly relevant in the context of the current opioid crisis. Medicinal and abused opioids act primarily on Mu Opioid Receptor (MOR) and we developed here a Cre mouse line to specifically target and manipulate MOR-expressing neurons. This resource is with huge potential for mapping, molecular characterization and functional studies of opioid-responsive neurons.