Opioid analgesic tolerance leads to dose-escalation in the clinic, dramatically reducing safety of chronic pain patients, due to deleterious CNS-mediated adverse consequences. Peripheral opioid delivery is a safer alternative as it provides effective local analgesia with limited central side-effects. However, tolerance also occurs peripherally, via mechanisms that remain unknown. Centrally, activation of the mu-opioid receptor (MOPr) by opioids induces release of platelet-derived growth factor-B (PDGF-B), and inhibition of PDGF receptor beta (PDGFRβ) prevents opioid tolerance. In the periphery, MOPr and PDGF-B are expressed in skin keratinocytes, and PDGFRβ is expressed in peripheral sensory neurons (PSNs), known to convey tolerance. Previous studies also showed that optogenetic stimulation of keratinocytes modulates PSNs via release of keratinocytes-derived factors. Thus, we hypothesized that mechanisms of peripheral opioid tolerance could involve keratinocytes and PDGFRβ signaling. We tested this hypothesis using mice behavioral pharmacology, optogenetics and in situ hybridization. We found that peripheral inhibition of PDGFRβ, using imatinib or a selective PDGFRβ antibody, could prevent tolerance from repeated intraplantar (i.pl.) morphine injections. Interestingly, PDGF-B was necessary and sufficient for peripheral morphine tolerance. In parallel, we discovered that repeated optogenetic activation of keratinocytes was sufficient to induce peripheral morphine tolerance which was reversed by inhibitors of PDGFRβ or of PDGF-B. Finally, we found that MOPr and PDGF-B were co-expressed in keratinocytes, and repeated i.pl. morphine increased PDGF-B expression in these cells. Together, these data show that peripheral tolerance to morphine involves activation of keratinocytes which may release PDGF-B and activate PDGFRβ signaling on PSNs. NIH NIDA R21 DA051636.