Electroacupuncture (EA) is widely used in clinical settings to reduce inflammatory pain. Islet-cell autoantigen 69 (ICA69) has been reported to regulate long-lasting hyperalgesia in mice. ICA69 knockout led to reduced protein interacting with C-kinase 1 (PICK1) expression and increased glutamate receptor subunit 2 (GluR2) phosphorylation at Ser880 in spinal dorsal horn. In this study, we evaluated the role of ICA69 in the antihyperalgesic effects of EA and the underlying mechanism through regulation of GluR2 and PICK1 in spinal dorsal horn. Hyperalgesia was induced in mice with subcutaneous plantar injection of complete Freund adjuvant (CFA) to cause inflammatory pain. Electroacupuncture was then applied for 30 minutes every other day after CFA injection. When compared with CFA group, paw withdrawal frequency of CFA+EA group was significantly decreased. Remarkable increases in Ica1 mRNA expression and ICA69 protein levels on the ipsilateral side were detected in the CFA+EA group. ICA69 expression reached the peak value around day 3. More importantly, ICA69 deletion impaired the antihyperalgesic effects of EA on GluR2-p, but PICK1 deletion could not. Injecting ICA69 peptide into the intrathecal space of ICA69-knockout mice mimicked the effects of EA analgesic and inhibited GluR2-p. Electroacupuncture had no effects on the total protein of PICK1 and GluR2. And, EA could increase the formation of ICA69-PICK1 complexes and decrease the amount of PICK1-GluR2 complexes. Our findings indicate that ICA69 mediates the antihyperalgesic effects of EA on CFA-induced inflammatory pain by regulating spinal GluR2 through PICK1 in mice.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.