Our current knowledge about sulfur(S) management by winter oilseed rape to satisfy the S demand of developing seeds is still scarce, particularly in relation to S restriction. Our goals were to determine the physiological processes related to S use efficiency that led to maintain the seed yield and quality when S limitation occurred at the bolting or early flowering stages. To address these questions, a pulse-chase (SO42-)-S-34 labeling method was carried out in order to study the S fluxes from uptake and remobilization at the whole plant level. In response of S limitation at the bolting or early flowering stages, the leaves are the most important source organ for S remobilization during reproductive stages. By combining S-34-tracer with biochemical fractionation in order to separate sulfate from other S-compounds, it appeared that sulfate was the main form of S remobilized in leaves at reproductive stages and that tonoplastic SULTR4-type transporters were specifically involved in the sulfate remobilisation in case of low S availability. In response to S limitation at the bolting stage, the seed yield and quality were dramatically reduced compared to control plants. These data suggest that the increase of both S remobilization from source leaves and the root proliferation in order to maximize sulfate uptake capacities, were not sufficient to maintain the seed yield and quality. When S limitation occurred at the early flowering stage, oilseed rape can optimize the mobilization of sulfate reserves from vegetative organs (leaves and stem) to satisfy the demand of seeds and maintain the seed yield and quality. Our study also revealed that the stem may act as a transient storage organ for remobilized S coming from source leaves before its utilization by seeds. The physiological traits (S remobilization, root proliferation, transient S storage in stem) observed under S limitation could be used in breeding programs to select oilseed rape genotypes with high S use efficiency.