The unicellular green alga Chlamydomonas reinhardtii reacts to sulfur (S) starvation with the increased expression of numerous genes. One gene which is induced in illuminated anaerobic S-deprived cells is the ferredoxin-5 gene (FDX5). To test FDX5 transcriptional regulation in aerobic cultures, we used a real-time PCR analysis and an artificial microRNA approach. We demonstrated that FDX5 gene is controlled by S deprivation independently of anoxia-treatment. The Ser/Thr kinase SNRK2.1 is necessary for expression of FDX5 during deprivation to S. Copper response regulator 1 (CRR1) is not involved in FDX5 up-regulation in S-deficient cells under aerobic conditions. Furthermore, expression of FDX5 is negatively regulated by nitric oxide (NO). Moreover, truncated hemoglobin 1 (THB1) underexpression resulted in the decrease in FDX5 transcript abundance in S-deficient cells under aerobic conditions. Together, our results imply that the FDX5 gene is controlled by NO in THB1-dependent pathway under conditions of depleted S supply.