We cloned the full-length mitogen activated protein kinase kinase 3 (MKK3) cDNA sequence using therapid amplification of cDNA ends method to understand the physicochemical and functional characteristics ofMKK3 in Fenneropenaeus chinensis. F. chinensis MKK3 was analyzed using a bioinformatics method to explorethe sequence homology of MKK3 genes from different species. MKK3 gene expression levels were determined indifferent tissues by quantitative real-time polymerase chain reaction analysis before and after exposure to ammonia-N stress. The full-length cDNA sequence of the F. chinensis MKK3 gene (FcMKK3) was 1434 bp long andcontained a 33 bp 5′-untranslated region (UTR), a 390-bp 3′-UTR, and a 1011-bp open reading frame that encoded336 amino acid residues, with an isoelectric point (PI) of 6.08 and molecular mass of 37.89 kD. The homologyanalysis revealed that the FcMKK3 amino acid sequence had highly similarity with MKK3 of other species, suchas 69% identity with Nasonia vitripennis MKK3 and 68% identity with Ceratitis capitata MKK3. The phylogeneticanalysis showed that FcMKK3 was in the same class with other arthropod MKK3 genes. The FcMKK3 genewas expressed in intestine, gill, stomach, heart, lymph, hepatopancreas, muscle, and hemocytes, with significantdifferences in tissue expression levels. Relative expression of FcMKK3 in muscle was the highest, followed by thegill. FcMKK3 expression in hemocytes was the lowest. FcMKK3 expression after exposure to ammonia-N stresswas initially upregulated in intestine, gill, stomach, and muscle and then downregulated, followed by upregulation.FcMKK3 expression reached the first peak at 6 h, 6 h, 6 h, and 3 h in intestine, gill, stomach, and muscle, respectively,which was 2.33-fold (P<0.01), 1.56-fold (P<0.01), 2.99-fold (P<0.01), and 1.56-fold (P<0.01) of the fourtissues in control animals, respectively. The second peak was reached at 96 h, 72 h, 72 h, and 48 h, which was2.49-fold (P<0.01), 2.34-fold (P<0.01), 2.36-fold (P<0.01), and 5.58-fold (P<0.01) more than those in the controlgroup, respectively. Relative FcMKK3 expression levels in the experimental group were higher than those in thecontrol group at all testing points. In contrast, relative FcMKK3 expression in heart, hepatopancreas, and hemocyteswas downregulated initially and then upregulated. FcMKK3 expression was the lowest at 3 h, 3 h, and 6 h,which was 0.56-fold (P<0.01), 0.26-fold (P<0.01), and 0.72-fold (P<0.01) of the values in the three tissues fromanimals in the control group, respectively. Then, FcMKK3 expression was upregulated and reached peaks at 48 h,6 h, and 24 h, which were 2.16-fold (P<0.01), 2.53-fold (P<0.01), and 1.19-fold (P<0.05) of the control groupvalues, respectively. In conclusion, relative FcMKK3 expression was upregulated significantly in intestine, gill,stomach, heart, hepatopancreas, muscle, and hemocytes compared with those in the control group after exposure toammonia-N stress and showed different expression profiles. These results suggest that FcMKK3 might play importantroles in the F. chinensis stress response.