Nitric oxide plays an important role in regulating adaption of the model alga Chlamydomonas reinhardtii to various environmental stresses. One response to abiotic stresses is the accumulation of protective molecules such as proline and putrescine. The NO treatment led to a significant accumulation of proline in cells. Quantitative real-time expression analysis of proline metabolic genes in NO-treated cells showed a prolonged upregulation of the gene encoding γ-glutamyl kinase 1 (GGK1) in the glutamate biosynthetic pathway. Furthermore, truncated hemoglobin 2 (THB2)-underexpressing strains with an enhanced endogenous NO demonstrated a higher proline content and GGK1 mRNA abundances than the wild type. In contrast, transcription of the gene encoding ornithine δ-aminotransferase in the ornithine pathway of proline biosynthesis decreased after treatment with NO. This suggests the predominance of the glutamate pathway over the ornithine pathway. We also found that the expression of the proline dehydrogenase gene encoding a key enzyme in proline catabolism was downregulated in NO-treated cells. Chlamydomonas reinhardtii exposed to exogenous NO also showed an increased ornithine decarboxylase 2 mRNA and putrescine content. Our findings indicate a clear link between changes in NO application and proline and putrescine content via transcriptional regulation of respective enzymes.