Siniperca chuatsi is an economically important freshwater fish species cultivated in China. Infectious spleen and kidney necrosis virus (ISKNV) is the main causative agent of disease in S. chuatsi and causes huge economic losses in aquaculture. Viruses depend on cellular metabolisms for rapid replication and proliferation, and metabolomics analyses have revealed that the carbon flux of glutamine metabolism increases in ISKNV-infected cells. Previously, ISKNV has been reported to up-regulate the expression of glutaminase (GLS) and glutamate dehydrogenase (GDH) genes in host cells. These enzymes mediate glutamine metabolism and are necessary for the production of high-titer ISKNV particles. However, the mechanism of ISKNV-induced glutamine metabolism remains unclarified. The silent mating type regulation 2 homolog 3 (SIRT3) is an NAD+ -dependent deacetylase that regulates cellular metabolic conditions and participates in biological activities including cell cycle, mitochondrial ageing, and energy production. SIRT3 has been widely reported to respond to nutrient deficiencies and participate in cancer progression by regulating glutamine metabolism. The metabolic status of virus-infected cells is very similar to that of cancer cells, suggesting that SIRT3 may play a regulatory role during ISKNV infection. To clarify the potential mechanistic link between ISKNV and glutamine metabolism, we analyzed cloned sequences of S. chuatsi SIRT3 and detected expression of SIRT3 in different tissues of S. chuatsi. Then, the time-dependent expression patterns of the S. chuatsi SIRT3 in vitro and in vivo after ISKNV infection were assessed using real-time fluorescent quantitative PCR (qRT-PCR) and western blotting. Finally, the effects of SIRT3 on glutamine metabolism and the proliferation of ISKNV in host cells were analyzed by regulating the expression and the activity of SIRT3. Results showed that the S. chuatsi SIRT3 encoded 449 amino acids that showed high conservation. Expression of SIRT3 in 12 tissues of S. chuatsi was detected by qRT-PCR, with the maximum expressions obtained from the hind kidney. Moreover, the expression of SIRT3 was lower in the spleen, the target organ of ISKNV infections. In vivo and in vitro infection tests results showed that ISKNV infection significantly increased the expression of SIRT3 in S. chuatsi spleen and brain cells and that the time-dependent expression pattern of SIRT3 in spleen and brain cells was similar. Subsequently, we found that the expression of GLS and GDH, the copy number of the ISKNV genome, and the titer of ISKNV significantly increased in ISKNV-infected cells treated with 7 μmol/L honokiol and significantly decreased in ISKNV-infected cells treated and transfected with 10 μmol/L 3-TYP and siRNA-SIRT3, respectively. From the results described above, ISKNV infection was shown to induce the reprogramming of glutamine metabolism via SIRT3, suggesting that SIRT3 may be an effective target for antiviral therapy.