Lung cancer is one of the leading causes of cancer-related deaths worldwide, with non-small cell lung cancer (NSCLC) being the most prevalent subtype. Gemcitabine is a primary clinical option for the treatment of NSCLC. Nevertheless, it encounters challenges including drug resistance and severe adverse effects. High expression of regulator of G protein signaling 2 (RGS2) is associated with poor prognosis in NSCLC. Usenamine A (UD32–3), a natural compound derived from lichen usnea longissimi, exerts anti-tumor activities in certain types of cancer cells, however, its underlying molecular mechanisms in NSCLC are largely unknown. In this study, we demonstrated for the first time that UD32–3 exerts anti-NSCLC activity by targeting RGS2, thereby suppressing Notch1 to induce autophagy and promoting reactive oxygen species (ROS)-mediated endoplasmic reticulum (ER) stress. Suppression of RGS2 inhibited cell growth by inducing ROS-mediated ER stress and Notch1-mediated autophagy in NSCLC, whereas its overexpression had the opposite effects. Additionally, combined therapy with UD32–3 and gemcitabine exerted synergistic anti-NSCLC activity. Our findings suggest that RGS2 is a promising therapeutic target for the treatment of NSCLC, and combined therapy with UD32–3 and gemcitabine might be an alternative therapeutic strategy for certain NSCLC patients. Keywords: NSCLC; RGS2; Usenamine a; Gemcitabine; Autophagy.