Opioid receptors play an important role in mediating the spinal analgesia. The mu-opioid receptor is the major target of opioid drugs widely used in clinics. However, the regulatory mechanisms of analgesic effect and tolerance for clinical mu-opioid receptor-targeting opioids remain to be fully investigated. Previous studies showed the interaction of delta-opioid receptor with mu-opioid receptor to form the mu-opioid receptor/delta-opioid receptor heteromers that could be processed in the degradation pathway after delta-opioid receptor agonist treatment. Here, we showed that clinical mu-opioid receptor-targeting opioids, morphine, fentanyl, and methadone, but not tramadol, caused mu-opioid receptor co-internalization with delta-opioid receptors in both transfected human embryonic kidney 293 cells and primary sensory neurons. Prolonged treatment of morphine led to mu-opioid receptor co-degradation with delta-opioid receptors. Furthermore, fentanyl and methadone, but not tramadol, induced the drug tolerance similar to morphine. Thus, the clinical mu-opioid receptor-targeting opioids including morphine, fentanyl, and methadone induce mu-opioid receptor co-internalization with delta-opioid receptors, which may be involved in the analgesic tolerance of these opioids.