AIM: To investigate the effect of electroacupuncture (EA) on the mitochondria-dependent apoptotic signaling pathway in the ciliary muscle of guinea pigs with negative lens-induced myopia (LIM). METHODS: Guinea pigs were randomly divided into normal control (NC) group, LIM group, LIM+SHAM acupoint (LIM+SHAM) group, and LIM+EA group. Animals in the NC group received no intervention, while those in other three groups were covered with -6.0 diopter (D) lenses on right eyes. Meanwhile, animals in the LIM+EA group received EA at Hegu (LI4) combined with Taiyang (EX-HN5) acupoints, while those in the LIM+SHAM group were treated at sham points. After treatments for 1, 2, and 4wk, morphological changes in ciliary muscles were observed with hematoxylin and eosin (H&E) staining and nick end labeling (TUNEL), and the expression of the mitochondrial apoptotic signaling pathway-related molecules in ciliary muscles was measured by real-time quantitative polymerase chain reaction (qPCR) and Western blot. Additionally, the adenosine triphosphate (ATP) contents were also determined in ciliary muscles. RESULTS: Axial length increased significantly in the LIM and LIM+SHAM groups and decreased in the LIM+EA group. The ciliary muscle fibers were broken and destroyed in both LIM and LIM+SHAM groups, whereas those in the LIM+EA group improved significantly. TUNEL assay showed the number of apoptotic cells increased in the LIM and LIM+SHAM groups, whereas reduced in the LIM+EA group. ATP contents showed a significant decrease in the LIM and LIM+SHAM groups, whereas increased after EA treatment. Compared with the NC group, the dynamin-related protein 1 (DRP1), Caspase3, and apoptotic protease activator 1 (APAF1) levels were significantly increased in the LIM group and decreased in the LIM+EA group. CONCLUSION: The results provide evidence of EA inhibiting the development of myopia by regulating the mitochondrial apoptotic signaling pathway.