ABSTRACT Mycobacterium tuberculosis is dependent on cysteine biosynthesis, and reduced sulfur compounds such as mycothiol synthesized from cysteine serve in first-line defense mechanisms against oxidative stress imposed by macrophages. Two biosynthetic routes to l -cysteine, each with its own specific cysteine synthase (CysK1 and CysM), have been described in M. tuberculosis , but the function of a third putative sulfhydrylase in this pathogen, CysK2, has remained elusive. We present biochemical and biophysical evidence that CysK2 is an S -sulfocysteine synthase, utilizing O -phosphoserine (OPS) and thiosulfate as substrates. The enzyme uses a mechanism via a central aminoacrylate intermediate that is similar to that of other members of this pyridoxal phosphate-dependent enzyme family. The apparent second-order rate of the first half-reaction with OPS was determined as k _max / K _s = (3.97 × 10 ³ ) ± 619 M ⁻¹ s ⁻¹ , which compares well to the OPS-specific mycobacterial cysteine synthase CysM with a k _max / K _s of (1.34 × 10 ³ ) ± 48.2. Notably, CysK2 does not utilize thiocarboxylated CysO as a sulfur donor but accepts thiosulfate and sulfide as donor substrates. The specificity constant k _cat / K _m for thiosulfate is 40-fold higher than for sulfide, suggesting an annotation as S -sulfocysteine synthase. Mycobacterial CysK2 thus provides a third metabolic route to cysteine, either directly using sulfide as donor or indirectly via S -sulfocysteine. Hypothetically, S -sulfocysteine could also act as a signaling molecule triggering additional responses in redox defense in the pathogen upon exposure to reactive oxygen species during dormancy.