Chlorella pyrenoidosa was exposed to nonylphenol (NP) to investigate the tolerance, antioxidant response, removal efficiency, and biodegradation mechanism. We conducted studies on algal biomass, chlorophyll a content, and photosynthetic activity, and found that C. pyrenoidosa exhibited a high tolerance even at 8 mg L−1 of NP. Changes in peroxidase (POD) and superoxide dismutase (SOD) activities indicated that the NP-induced oxidative stress caused oxidant damage, which increased the malondialdehyde (MDA) content. After culturing for 120 h, the NP removal efficiency of C. pyrenoidosa was 89%, 59%, 49%, and 48% in the 2, 4, 6, and 8 mg L−1 treatment groups, respectively. Degradation intermediates determined by GC–MS suggested that the biodegradation of NP in C. pyrenoidosa originated from the long alkyl chain. In addition, transcriptome analysis indicated that NP affected photosynthesis, antioxidase, and oxidoreductase activity-related genes. In summary, our results indicated that C. pyrenoidosa is a species that exhibits high tolerance and biodegradation capacity toward NP.