Mirror-image pain (MIP), which occurs along with complex regional pain syndrome, rheumatoid arthritis and chronic migraine, is characterized by increased pain sensitivity of healthy body regions other than the actual injured or inflamed sites. A high level of peripheral inflammation may activate central or peripheral glia, triggering mirror-image pain. However, which receptors mediate inflammatory signals to contribute glial activation remains unclear. Intraplantarly injecting mice with 5-hydroxytryptamine (5-HT) or acidic buffer (proton) caused only unilateral hyperalgesia, but co-injection of 5-HT/acid induced bilateral hyperalgesia (MIP). Blocking 5-HT3 or acid-sensing ion channel 3 (ASIC3) abolished satellite glial activation, inhibiting MIP. Interestingly, intraplantar administration of a 5-HT3 agonist induced MIP, and 5-HT3–mediated MIP can be reversed by a 5-HT3 antagonist or an ASIC3 blocker. Similar results were found using a ASIC3 agonist. Furthermore, 5-HT3 was observed to co-localize with ASIC3 in DRG neurons; 5-HT3 activation-induced an increase in intracellular calcium that was inhibited by an ASIC3 blocker and vice versa. A cross-talk between 5-HT3 and ASIC3 mediates satellite glial activation, thereby triggering mirror-image pain.