Sulfate and nitrate assimilatory pathways are well coordinated so that the absence of one element affects the other pathway. To date, their assimilations have been established. However, the interactions between sulfate and nitrate assimilation as well as the regulatory mechanisms were still under debate. Thus, to address those issues, eight-week-old plants grown under hydroponic were deprived of sulfur (S) or nitrogen (N) for 3 days, and then the plants were externally supplied with 1 mM different N- or S-assimilates (glutamine [Gln], glutathione [GSH], glutamate [Glu], proline [Pro] or cysteine [Cys]), nitrate or sulfate itself. The sampling was done before treatment (day 0), after 3 days of N- or S-deprivation (day 3), and after 3 more days of N- or S-assimilates feeding (day 6). After sampling, nitrate and sulfate assimilatory enzymes, relevant gene expressions and the content of N- and S-assimilates were measured at day 0, 3 and 6, respectively. S-deprivation for 3 days significantly increased the activity of adenosine 5´-triphosphate sulfurylase (ATPs) and adenosine 5´-phosphosulfate reductase (APR). However, O-acteylserine(thiol)lyase (OASTL), nitrate reductase (NR) and glutamine synthetase (GS) were dramatically decreased. The products derived from sulfate or nitrate assimilation such as Cys, GSH and protein were decreased upon S depletion, but an accumulation in amino acids was observed. The expression of sulfate transporter genes group 1 and 4 were strongly induced in response to S-deprivation. However, NRT1;1 expression was depressed. N-deprivation significantly repressed the level of Sultr1;1 and Sultr4;1. Inversely, it de-repressed the expression of Sultr1;2 and Sultr4;2. The effects of the feeding of possible regulatory metabolites under N- or S-deprived condition on N- and S-assimilatory enzyme activities and relevant gene expressions were examined for 3 days. The resupply of sulfate tended to recover both sulfate and nitrate assimilations. Meanwhile Cys exhibited a feedback effect on the expression of sulfate transporter genes group 1, the activity of ATPs and APR. In comparison with other metabolites conducted in current study Cys appeared to more effectively influence sulfate assimilation. Moreover, Cys induced the expression of NRT1;1 and the activity of GS, but diminished NR activity. Under N-deprived condition, nitrate re-feeding was likely to recover both nitrate and sulfate assimilations. N-deprivation significantly decreased the activity of NR, APTs, APR and OASTL, while induced GS activity. In terms of N- or S-assimilates feeding, it was found that Gln adding repressed the expression of NRT1;1 and the activity of GS. On the other hand, the addition of Gln decreased the activity of ATPs and APR, but increase OASTL activity. By contrast, other relevant metabolites less affected the assimilation of nitrate or sulfate under N-deprived condition. Remarkably, Pro appeared to influence both assimilations. It profoundly repressed the expression of NRT1;1 and ATPs activity, but increased NR activity. The assembled data indicated that S- and/or N-deprivation severely modified the assimilatory pathway of sulfate and nitrate with coordination of each other, and that Cys in S-deprivation while Gln in N-deprivation played more important roles than other related assimilates in modulating sulfate and/or nitrate assimilation under S- and N-derived condition in Brassica napus L.