Plant S deficiency is common, but the role of S-containing amino acids such as cysteine in plant S uptake is unknown. We applied 14C-, 35S-, 13C-, and 15N-labelled cysteine to wheat and oilseed rape rhizospheres and traced the plants’ elemental uptake. Both plants absorbed 0.37–0.81% of intact cysteine after 6 h with no further increase after 24 h. They absorbed 1.6–11.5% 35S and 12.3–7.6% 15N from cysteine after 24 h and utilised SO42− as their main S source (75.5–86.4%). Added and naturally occurring cysteine-S contributed 5.6 and 1.1% of total S uptake by wheat and oilseed rape, respectively. Cysteine and inorganic S derived from cysteine contributed 24.5 and 13.6% of uptake for wheat and oilseed rape, respectively, after 24 h. Oilseed rape absorbed ~10-fold more S from cysteine and SO42− than did wheat. The highest absorption of free cysteine should be in the organic-rich soil patches. Soil microorganisms rapidly decomposed cysteine (t1/2 = 1.37 h), and roots absorbed mineralised inorganic N and S. After 15 min, 11.7–14.3% of the 35S-cysteine was retained in the microbial biomass, while 30.2–36.7% of the SO42− was released, suggesting that rapid microbial S immobilisation occurs after cysteine addition. Plants acquire N and S from cysteine via unidirectional soil-to-root nutrient flow, and cysteine is an important S source for plants.