Abstract The de novo pyrimidine biosynthetic pathway is an inducible cellular program which allows the rapid synthesis of pyrimidine nucleotides in proliferating cells, and the final rate-limiting step is catalyzed by cytidine triphosphate synthase (CTPS1 or CTPS2). A CTPS1 loss-of-function mutation in humans has illustrated the essential and non-redundant role of CTPS1 in activated lymphocyte proliferation. Therefore, selective CTPS1 inhibition is an attractive approach to specifically target pathological immune activity in autoimmune and inflammatory disease. Using cryo-EM and structure-based drug design, we have identified a series of potent, orally bioavailable, CTPS1-specific inhibitors, exemplified by the compound NTX-871. In-vitro, NTX-871 significantly inhibits the proliferation of T-cell derived lines and activated primary T-cells. These inhibitory effects are reversed by the addition of exogenous cytidine, illustrating the specificity of NTX-871 for CTPS1 and the de novo pyrimidine synthesis program. NTX-871 was also tested in several mouse in-vivo PD and autoimmune disease models. Oral dosing of NTX-871 significantly reduced T-cell proliferation in an ovalbumin-induced T-cell proliferation model, and QD or BID dosing of NTX-871 significantly improved disease phenotypes in models of delayed-type hypersensitivity, collagen-induced arthritis, and experimental autoimmune encephalomyelitis.