Main conclusion: Truncated hemoglobin 2 is involved in fine-tuning of PSR1-regulated gene expression during phosphorus deprivation. Abstract: Truncated hemoglobins form a large family found in all domains of life. However, a majority of physiological functions of these proteins remain to be elucidated. In the model alga Chlamydomonas reinhardtii, macro-nutritional deprivation is known to elevate truncated hemoglobin 2 (THB2). This study investigated the role of THB2 in the regulation of a subset of phosphorus (P) limitation-responsive genes in cells suffering from P-deficiency. Underexpression of THB2 in amiTHB2 strains resulted in downregulation of a suite of P deprivation-induced genes encoding proteins with different subcellular location and functions (e.g., PHOX, LHCSR3.1, LHCSR3.2, PTB2, and PTB5). Moreover, our results provided primary evidence that the soluble guanylate cyclase 12 gene (CYG12) is a component of the P deprivation regulation. Furthermore, the transcription of PSR1 gene for the most critical regulator in the acclimation process under P restriction was repressed by nitric oxide (NO). Collectively, the results indicated a tight regulatory link between the THB2-controlled NO levels and PSR1-dependent induction of several P deprivation responsive genes with various roles in cells during P-limitation.