Self-injurious behavior (SIB) is commonly observed in patients with neuropsychiatric disorders, as well as nonclinical populations with stress related mental health problems. However, the exact circuitry mechanisms underlying SIB have remained unclear. Here we established a rat model of SIB with bilateral injection of muscimol into entopeduncular nucleus (EP). Following the muscimol injection, the male rats exhibited stereotypic self-biting behavior that lasted for hours and often resulted in wounds of various severities, in a dose-dependent manner. The SIB was associated with elevated level of serum corticosterone, and could be exacerbated or alleviated by enhancing or inhibiting the corticosterone signaling, respectively. Activity mapping using c-fos immunostaining, combined with connectivity mapping using herpes simplex virus-based anterograde tracing from the EP and pseudorabies virus-based retrograde tracing from the masseter muscle, revealed the potential involvement of many brain areas in SIB. In particular, the lateral habenula (LHb) and the ventral tegmental area (VTA), the two connected brain areas involved in stress response and reward processing, showed significant increase in neuronal activation during SIB. Furthermore, suppressing the LHb activity or modulating the GABAergic transmission in the VTA could significantly reduce the occurrence of SIB. These results demonstrate the importance of the stress hormone signaling and the LHb-VTA circuit in modulating SIB resulted from EP malfunction, and suggest potential targets for therapeutic intervention of SIB and related disorders.SIGNIFICANCE STATEMENTSelf-injurious behavior (SIB) occurs in about 4% of the general population, with substantially higher occurrence among adolescents and patients of neuropsychiatric disorders. Stress has been linked to the occurrence of SIB, yet the underlying mechanisms have remained unclear. Using a rat model of SIB induced by disruption of activity in the entopeduncular nucleus (EP), we found that the behavior is regulated by stress and linked to corticosterone signaling. Viral tracing and c-fos immunostaining revealed the involvement of various subcortical areas, especially the EP-lateral habenula (LHb)-ventral tegmental area (VTA) circuit, in SIB. Furthermore, regulating activity in the LHb or the VTA alleviates SIB. These results may have implications in the development of new strategies to treat SIB.