The cysteine biosynthetic pathway is absent in humans but essential in microbial pathogens, suggesting that it provides potential targets for the development of novel antibacterial compounds. CysK1 is a pyridoxalphosphate-dependent O-acetyl sulfhydrylase, which catalyzes the formation of l-cysteine from O-acetyl serine and hydrogen sulfide. Here we report nanomolar thiazolidine inhibitors of Mycobacterium tuberculosis CysK1 developed by rational inhibitor design. The thiazolidine compounds were discovered using the crystal structure of a CysK1-peptide inhibitor complex as template. Pharmacophore modeling and subsequent in vitro screening resulted in an initial hit compound 2 (IC50 of 103.8 nM), which was subsequently optimized by a combination of protein crystallography, modeling, and synthetic chemistry. Hit expansion of 2 by chemical synthesis led to improved thiazolidine inhibitors with an IC50 value of 19 nM for the best compound, a 150-fold higher potency than the natural peptide inhibitor (IC50 2.9 μM).