Hydrogen sulfide (H2S) is an important biological mediator and has been at the center of a rapidly expanding field focused on understanding the biogenesis and action of H2S as well as other sulfur-related species. Concomitant with this expansion has been the development of new chemical tools for H2S research. The use of H2S-selective fluorescent probes that function by H2S-mediated reduction of fluorogenic aryl azides has emerged as one of the most common methods for H2S detection. Despite this prevalence, the mechanism of this important reaction remains under-scrutinized. Here we present a combined experimental and computational investigation of this mechanism. We establish that HS(-), rather than diprotic H2S, is the active species required for aryl azide reduction. The hydrosulfide anion functions as a one-electron reductant, resulting in the formation of polysulfide anions, such as HS2(-), which were confirmed and trapped as organic polysulfides by benzyl chloride. The overall reaction is first-order in both azide and HS(-) under the investigated experimental conditions with ΔS(⧧) = -14(2) eu and ΔH(⧧) = 13.8(5) kcal/mol in buffered aqueous solution. By using NBu4SH as the sulfide source, we were able to observe a reaction intermediate (λ(max) = 473 nm), which we attribute to formation of an anionic azidothiol intermediate. Our mechanistic investigations support that this intermediate is attacked by HS(-) in the rate-limiting step of the reduction reaction. Complementing our experimental mechanistic investigations, we also performed DFT calculations at the B3LYP/6-31G(d,p), B3LYP/6-311++G(d,p), M06/TZVP, and M06/def2-TZVPD levels of theory applying the IEF-PCM water and MeCN solvation models, all of which support the experimentally determined reaction mechanism and provide cohesive mechanistic insights into H2S-mediated aryl azide reduction.