S-Ribosylhomocysteinase (LuxS) catalyzes the cleavage of the thioether bond in S-ribosylhomocysteine to produce L- homocysteine and 4,5-dihydroxy-2,3-pentanedione, the precursor of type II bacterial quorum sensing autoinducer. This work carried out extensive mechanistic studies of the LuxS reaction. The native metal cofactor of LuxS was identified as ferrous ion, instead of previously reported zinc ion, with a potential catalytic role. Substantial evidence was provided for the internal redox reaction, which comprised two consecutive carbonyl migration steps followed by β-elimination. Three LuxS activity assays were developed and greatly facilitated the mechanistic investigations of LuxS. Two classes of LuxS inhibitors were designed based on metal chelation and catalytic mechanism, respectively. They encouraged future development of LuxS inhibitors as novel antibacterial agents and helped probe the catalytic mechanism of LuxS.