The present study was conducted to validate the acetylation site of STAT3 and its relationship with SIRTs deacetylase family in Pelteobagrus fulvidraco. The STAT3 overexpression plasmids containing Flag tag and the SIRTs overexpression plasmids containing HA and GFP tags were constructed, and mutants were constructed for the possible acetylated sites of STAT3. Then, STAT3 was transfected and the mutants, STAT3 and SIRTs were co-transfected into HEK 293T cells, and the detection was carried out by immunoblotting, immunoprecipitation and immunofluorescence techniques. The results showed that STAT3 is a cytoplasmic protein. Compared with wild-type STAT3, the acetylation level of mutant K685R was significantly reduced. The acetylation levels of mutants K49R, K87R, K680R, K712R and K714R had no significant changes compared with wild-type STAT3. The immunoprecipitation and immunofluorescence showed that SIRT2 and SIRT7 interacted with STAT3 and catalyzed the deacetylation of STAT3, while SIRT5 did not interact with STAT3, and there was no significant change in acetylation level. The study revealed the acetylation site of STAT3 and the relationship between STAT3 and SIRTs in P. fulvidraco.