Nitrite is a harmful substance in aquaculture, and has a serious impact on the survival of the Chinese soft-shelled turtle, Pelodiscus sinensis. However, the cellular responses of P. sinensis to nitrite stress have not yet been investigated. The present study showed that nitrite led to a decrease in hemoglobin content and an increase in methemoglobin content in the blood, thus reducing the oxygen-carrying capacity of blood in P. sinensis. Nitrite also affects the antioxidant system of the liver and leads to lipid peroxidation. In addition, nitrite caused immune responses, including a decrease in lysozyme content and an increase in total complement activity, interleukin-6, and heme oxygenase concentrations in the serum. Additionally, the terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay showed that apoptosis occurred in liver cells. Finally, a comparative transcriptome analysis was conducted. A total of 573 differentially expressed genes and 15 significantly enriched KEGG pathways were identified. Among them, the glutathione S-transferase omega 1 (GSTO1) gene may relieve nitrite-induced oxidative damage in P. sinensis by participating in a variety of redox-related pathways, while the PPAR signaling pathway has been proposed to play an important regulatory role in lipid metabolism and immune responses. The present study comprehensively explored the cellular responses of P. sinensis to nitrite stress and provided guidance for future studies.