Sulfur is essential for plant growth and development, and the molecular systems for maintaining sulfur and thiol metabolism are tightly controlled. From a biochemical perspective, the regulation of plant thiol metabolism highlights nature’s ability to engineer pathways that respond to multiple inputs and cellular demands under a range of conditions. In this review, we focus on the regulatory mechanisms that form the molecular basis of biochemical sulfur sensing in plants by translating the intracellular concentration of sulfur-containing compounds into control of key metabolic steps.These mechanisms range from the simple (substrate availability, thermodynamic properties of reactions, feedback inhibition, and organelle localization) to the elaborate (formation of multienzyme complexes and thiol-based redox switches). Ultimately, the dynamic interplay of these regulatory systems is critical for sensing and maintaining sulfur assimilation and thiol metabolism in plants.