Transcranial focused ultrasound (tFUS) is regarded as a promising non-invasive stimulation tool for modulating brain circuits. The aim of this study is to explore the feasibility of tFUS stimulation for analgesia applications.50 µl of 3% formalin solution was injected into the rat's left hindpaw to build a pain model, and then the local field potential (LFP) activities of the dorsal horn were tracked after a recording electrode was placed in the spinal cord. Rats were randomly divided into two groups: control group and tFUS group. At the 30th minute after formalin injection, tFUS (US-650 kHz, PD = 1 ms, PRF = 100 Hz, 691 mW/cm2) was conducted to stimulate the periaqueductal gray (PAG) for 5 minutes (on 5 s and off 5 s) in the tFUS group, but there was no treatment in the control group. In addition, the analgesia mechanism (LFP recording from the PAG) and safety assessment (histology analysis) were carried out.The tFUS stimulation of the PAG can suppress effectively the nociceptive activity generated by formalin. The findings of the underlying mechanism exploration indicated that the tFUS stimulation was able to activate the PAG directly without causing notable temperature change and tissue injury.The results illustrated that the tFUS stimulation of the PAG can achieve the effect of analgesia.This work provides new insights into the development of non-invasive analgesic technology in the future.