We cloned the full-length mitogen activated protein kinase kinase 3 (MKK3) cDNA sequence using the rapid amplification of cDNA ends method to understand the physicochemical and functional characteristics of MKK3 in Fenneropenaeus chinensis. F. chinensis MKK3 was analyzed using a bioinformatics method to explore the sequence homology of MKK3 genes from different species. MKK3 gene expression levels were determined in different 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 and contained a 33 bp 5''-untranslated region (UTR), a 390-bp 3''-UTR, and a 1011-bp open reading frame that encoded 336 amino acid residues, with an isoelectric point (PI) of 6.08 and molecular mass of 37.89 kD. The homology analysis revealed that the FcMKK3 amino acid sequence had highly similarity with MKK3 of other species, such as 69% identity with Nasonia vitripennis MKK3 and 68% identity with Ceratitis capitata MKK3. The phylogenetic analysis showed that FcMKK3 was in the same class with other arthropod MKK3 genes. The FcMKK3 gene was expressed in intestine, gill, stomach, heart, lymph, hepatopancreas, muscle, and hemocytes, with significant differences in tissue expression levels. Relative expression of FcMKK3 in muscle was the highest, followed by the gill. FcMKK3 expression in hemocytes was the lowest. FcMKK3 expression after exposure to ammonia-N stress was 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 four tissues in control animals, respectively. The second peak was reached at 96 h, 72 h, 72 h, and 48 h, which was 2.49-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 control group, respectively. Relative FcMKK3 expression levels in the experimental group were higher than those in the control group at all testing points. In contrast, relative FcMKK3 expression in heart, hepatopancreas, and hemocytes was 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 from animals 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 group values, 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 to ammonia-N stress and showed different expression profiles. These results suggest that FcMKK3 might play important roles in the F. chinensis stress response.