ADP-ribosylation factor 1 (Arf1) is a crucial regulator in vesicle-mediated membrane trafficking and involved in the activation of signaling molecules. However, the molecular mechanisms underlying Arf1-dependent cancer development and progression are far from understood. We report here that Arf1 is the most amplified gene in Arf gene family in breast cancer, and knocking it down leads to an induction of autophagy via the AMPK pathway. To effectively target Arf1, we rationally designed and synthesized constrained ATC-based (4-amino-(methyl)-1,3-thiazole-5-carboxylic acid) γ-dipeptides, which can block Arf1 activation more efficiently than other commercially available Arf1 inhibitors through targeting the Arf1-GDP/ARNO complex at the Golgi. The anticancer effects of these γ-dipeptides were evaluated in triple native breast cancer MDA-MB-231 cells and their bone-seeking and lung-seeking sublines, showing reduced cell viability along with significant increased autophagy following treatment in both 2D and 3D cell cultures. Among these new inhibitors, the γ-dipeptide 10b displayed strongest cytotoxicity. The orthotopic NSC mouse model for breast cancer further showed the γ-dipeptides 10b exhibited a superior anticancer effects by inducing autophagic cell death in breast tumor, with no significant systemic toxicity. These findings reveal that Arf1-targeting γ-dipeptides developed by our collaborative team may represent a promising targeted therapeutic to improve treatment of breast cancer. Citation Format: Leilei He, Alain Chavanieu, Yen Vo-Hoang, Yong Teng. Novel Arf1-targeting ã-dipeptides counteract triple negative breast cancer by inducing autophagic death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1229.