Abstract Background: Tim-3 (T-cell immunoglobin and mucin-domain containing-3) has emerged as a promising novel immune checkpoint target playing non-redundant and synergistic roles with PD-1 in triggering T cell exhaustion, however, the complexity of Tim-3-interacting ligands (Gal-9, Ceacam1 and others) has posed a challenge to reconstitute functional Tim-3-targeting immune checkpoint blockers (ICBs) cellular screening system. Materials and methods: To address this challenge, we have engineered Tim-3 Advanced Genomic Manipulated human Jurkat T IL-2-Luc2P reporter cells (Tim-3 AGM T cells) via multiple steps of electroporation-mediated genomic manipulation, and validated the functionality of this reconstituted Tim-3 immune checkpoint cellular system using IL-2-Luc2P reporter assay and IL-2 ELISA following TCR and CD28 engagement. Results: We found that anti-CD3/CD28-induced T cell activation in the Tim-3 AGM T cells as assessed by IL-2-Luc2P reporter was significantly suppressed (~80%) after over-expression of Gal-9 but not the empty vector control, validating functionality of the reconstituted Tim-3/Gal-9 immune checkpoint complex. Conclusions: Such an innovative reconstituted functional Tim-3/Gal-9 immune checkpoint reporter cell line can enable high through-put screening for novel Tim-3 ICBs including small or large molecule therapeutics and following in-depth mechanistic studies. Citation Format: Fang Zhang, Pei Wu, Jun Liu, Baiqiu Wang. Reconstituted functional Tim-3/Gal-9 immune checkpoint cell line to screen for novel Tim-3 ICBs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3258.