OBJECTIVE To observe the effect of electroacupuncture(EA) on the pain behavior and prostaglandin E2 (PGE2), calcitonin gene-related peptide (CGRP) and substance P (SP) in the spinal cord dorsal horn and dorsal root ganglia (DRG) of rats with knee osteoarthritis (KOA), so as to explore the mechanisms of EA underlying improvement of chronic pain in KOA rats. METHODS Thirty-two female SD rats were randomly divided into blank group, control group, model group and EA group, with 8 rats in each group. Rats in the control group were injected with 50 μL of 0.9% sodium chloride solution into the left knee joint cavity, and rats in the model and EA groups were injected with 50 μL of Monosodium iodoacetate in the left knee joint. EA(2 Hz/100 Hz, ＜2 mA) was applied to left "Yanglingquan"(GB34) and "Neixiyan" (EX-LE4) for 15 min, once daily, 5 days a course with a total of 2 courses. Paw withdrawal latency (PWL) and mechanical pain threshold (PWT) were tested by Plantar Test and Von Frey, separately. After the last pain test, the contents of PGE2, CGRP and SP in the left lumbar (L) 3-L5 DRG and L3-L5 spinal dorsal horn were detected by ELISA. RESULTS Compared with the blank group, the PWL and PWT of the rats in the model group were significantly decreased (P<0.01), and the contents of PGE2, CGRP and SP in the DRG and spinal dorsal horn were significantly increased (P<0.01, P<0.05). There were no statistically significant differences in PWL, PWT, contents of PGE2, CGRP and SP in DRG and spinal dorsal horn between the blank group and the control group (P>0.05). Compared with the model group, the PWL and PWT of rats in the EA group were significantly increased (P<0.01), and the contents of PGE2, CGRP and SP in the DRG and spinal dorsal horn were significantly decreased (P<0.01, P<0.05). CONCLUSIONS EA of GB34 and EX-LE4 can reduce the levels of pain-related factors PGE2, CGRP and SP in the DRG and spinal dorsal horn, thereby relieving spinal hyperalgesia in rats with KOA.