Abstract: This article primarily investigates the role of the neural circuit from lateral hypothalamic (LH) glutamatergic neurons to the rostromedial tegmental nucleus (RMTg) in defensive behaviors in mice. Adult male vesicular glutamate transporter 2 (Vglut2)-Cre mice (8-10 weeks old) were randomly divided into a mCherry control group and a channelrhodopsin-2 (ChR2) experimental group, who received injections of AAV2/9-EF1α-DIO-mCherry and AAV2/9-EF1α-DIO-ChR2-mCherry virus into the LH, respectively. Optical fibers were implanted in the RMTg of both groups. After three weeks of recovery from surgery and sufficient viral expression, behavioral experiments were conducted, including the open field test, real-time place preference (RTPP) test, and hiding box test. After the experiment, ascertain the extent of viral infection and optical fiber positions in the mice. Immunohistochemistry was carried out to detect c-Fos expression in the RMTg to validate the activation of the pathway from LH glutamatergic neurons to the RMTg upon 473 nm blue laser stimulation. The results showed that c-Fos expression in the RMTg was elevated in ChR2-expressing mice following blue laser illumination, confirming successful pathway activation. During a 473 nm blue laser stimulation, compared to the mCherry group, mice in the ChR2 group exhibited a notable increase in velocity in the open field test, indicating an escape behavior. In the RTPP test, the time spent on the preferred side reduced, indicating avoidance behavior. Additionally, the latency to enter the hiding box significantly shortened, reflecting hiding behavior in the hiding box test. This research indicates that the activation of the projections from LH glutamatergic neurons to the RMTg promotes defensive behaviors in mice. All experimental procedures were approved by the Committee on the Ethics of Animal Experiments of the School of Basic Medical Sciences, Fudan University (identification number: 20250427-001).