Herbicides present in the environment induce oxidative stress in plants. We investigated the roles of exogenous NO-regulated chlorophyll synthesis, antioxidant enzyme activity and gene expression in herbicide-treated unicellular green algae Chlorella vulgaris. Atrazine (100 microg/L) or glufosinate (10 mg/L) treatment alone or in combination with 20 microM sodium nitroprussiate (SNP, 10 microM with glufosinate) was administered to algae for a short time period of 48 h to observe changes in physiology and gene transcription and expression. Supplementation of atrazine or glufosinate with low SNP significantly reduced H2O2, reactive oxygen species (ROS) and malondialdehyde (MDA) induction by herbicides. Supplementation also increased chlorophyll content and antioxidant enzymes, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity, as compared to herbicide treatment alone. This trend suggests an effect of NO on the scavenging of ROS. Furthermore, the expression of photosynthesis genes (psbC, psaB, chlB and rbcL) was also upregulated by supplementation of low SNP, thus maintaining the normal photosynthetic function. However, high concentration of SNP (100 microM) in combination with herbicides aggravated damage to algae, including increases in H2O2, ROS and MDA and decreases in chlorophyll content, antioxidant enzyme activity and photosynthesis genes transcription. 2-Phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO), the NO scavenger, was also examined in this study; the results showed that PTIO could neutralise the effect of low SNP. Data also showed that an exogenous supply of NO protects Chlorella vulgaris against the toxicity of herbicides by protecting against oxidant substances and increasing the transcription of related genes.