The effect of periaqueductal gray (PAG) injection of neurotensin (NT), anti-NT serum (ANTS), and naloxone (Nx) on both the pain threshold and electroacupuncture (EA) analgesia in rat was investigated in this study. The potassium iontophoresis-induced tail-flick was used to measure the pain threshold. NT administration induced an increase in pain threshold and enhanced EA analgesia. Injection of ANTS reduced the pain threshold significantly and diminished the effect of EA analgesia. Furthermore, pre-injection of Nx into PAG could weaken analgesia effect of NT and NT-EA analgesia. These results indicate that NT in PAG is involved in pain modulation and plays a role in EA analgesia. The effect of NT may be partly conducted by endogenous opiate peptides.