Nitric oxide (NO) is a signal in the modulation of acclamatory responses to stress in plants. This study has investigated the metabolic shift in the acclimation of a green alga Chlamydomonas reinahrdii to NO stress. On exposure to 0.1 mM SNAP, a NO donor, causes a transient inhibition in photosysnthesis after 1 h, followed by a fast recovery. The RNA-seq results discover that NO enhances the catabolism of cysteine, threonine, aromatic amino acids, and branched-chain amino acids to other metabolites for the synthesis of functional compounds. NO also induces the endosomal protein trafficking system in association with the ubiquitinylation machinery (ubiquition, SNARE, and autophagy) for the degradation of sequestered damaged proteins to generate small molecules for re-synthesis of macromolecules, and triggers the protein modification system (heat proteins). The antioxidant defense system (VTC2, APX, MDAR, DHAR, GR, GPXH/GPX5, GSTS1, and GSTS2) and redox state (AsA/DHA and GSHH/GSSG) are activated by NO. Two PsbP-like proteins and LHCBM9 are induced for the acclimayion to NO stress. Besides, THB, FLVb, and CYP55B1 are induced to reduce NO level for NO detoxification, allowing the acclimation to NO stress. NO significantly induced NADPH oxidase, RBOL2, but slightly decreased RBOL1 expression. Unfolded protein response (UPR) in response to ER stress was inhibited by NO stress. Using RBOL2 mutant, the UPR was induced and the glutathione synthesis and redox state was inhibited. This finding demonstrates that NADPH oxidase (RBOL2)-mediated signaling pathway is involved in the NO-induced inhibition of ER stress and the induction of glutathione availability while other NO-induced metabolisms are under control by other signaling pathway. Our analyses provide a valuable tool for selecting genetic modification targets to understand acclamatory mechanisms to NO stress in Chlamydomonas.