Thiamin phosphate synthase (ThiE) catalyzes the formation of thiamin phosphate (thiamin-P) from 4-amino-5-(hydroxymethyl)-2-methylpyrimidine pyrophosphate (HMP-PP) and 5-(hydroxyethyl)-4-methylthiazole phosphate (Thz-P). This reaction is the penultimate step in the biosynthesis of thiamin pyrophosphate, the active form of vitamin B1. According to available genomic data, all organisms that are capable of synthesizing thiamin have some form of this coupling enzyme. In regard to the enzymatic mechanism, some previous structural and biochemical data support the notion that pyrophoshate (PPi) dissociates from HMP leading to a carbocation which is rapidly trapped by Thz-P to form Thiamin-P. We offer direct evidence that the reaction proceeds via this SN1-type mechanism. It was shown that wild-type ThiE from B. subtilis exhibits a pre-steady state burst of product formation. An extensive transient state kinetic characterization resulted in the identification of the rate-limiting step involving the burst. This rate increased over 15-fold when a methoxy substituent was present at C2 of HMP-PP (Bacimethrin-PP) showing that the electron donating group had a direct effect on the rate of the chemical reaction. Observation of tyrosine fluorescence quenching in a stopped-flow apparatus enabled an accurate measurement of most of the microscopic rate constants in the kinetic pathway, leading to a detailed understanding of the ThiE Mechanism. The overall limiting step of steady state turnover is due to slow thiamin-P release.