During sulfur (S) deprivation, Chlamydomonas reinhardtii mediates a suite of specific responses to efficiently acquire S from various sources and remodel primary metabolism. Although the truncated hemoglobin 1 (THB1)-dependent regulation of a subset of S limitation-responsive genes has been elucidated, the mechanism through which THB1transcription is triggered in S-deprived cells is not known. Here, we show that signaling proteins and regulators associated with S deprivation specific responses did not control THB1 expression. Following S depletion, rapid increase in nitrite concentrations is essential for THB1upregulation. These changes are consistent with NO accumulation. Increased nitrite, NO and THB1mRNA levels were not observed in mutant cells without diaphorase-NR activity. Our findings suggest that nitrate reductase - nitric oxide-forming nitrite reductase system plays a positive role in THB1 transcription via generation of NO from nitrite in S-starved cells. NR-dependent NO production may be regarded as an early trigger, which contributes to Chlamydomonas adaptability to nutrient stresses.