Abstract: To study the sequence characteristics, the gene expression patterns under salinity stress and the targeted regulation of related miRNAs of NHE3 and NKAα1a in cobia, Rachycentron canadum, the full-length cDNA sequences of NHE3 and NKAα1a were cloned by rapid amplification of cDNA ends; the tissue-specific and salinity-adaptive expression patterns of NHE3 and NKAα1a were analyzed by real-time quantitative PCR; the double-luciferase reporter assay was used to detect the targeted regulatory relationship between NHE3 and NKAα1a and related miRNAs. The open reading frames of cobia NHE3 and NKAα1a were 2718 bp and 3075 bp in length, encoding 905 and 1024 amino acids, respectively. NHE3 and NKAα1a were expressed in all detected tissues, including gill, intestine, and heart, among which the highest expression abundance was found in gill. With the increase of salinity, the expression of NHE3 in gill decreased gradually, and there were significant differences between low-salt and high-salt adaptation. With the increase of salinity, the expression level of NKAα1a showed different trends, and it was significantly up-regulated in gill and intestine after being challenged by low-salt and high-salt conditions, while its expression level was significantly down-regulated in kidney in high-salt environment. In different salinity adaptation processes, the highest expression levels of NHE3 and NKAα1a were all found in the gill. When NHE3-pmirGLO-WT was co-transfected with miR-1335-3p, the relative luciferase activity decreased compared with the control group, and there was a very significant difference, and the similar results were found when NKAα1a-pmirGLO-WT was co-transfected with miR-1788-3p and mimic NC (control). The results of the double-luciferase reporter assay suggested that miR-1335-3p and miR-1788-3p could bind to NHE3 and NKAα1a 3′-UTR sequences, respectively, and downregulate their mRNA expression levels; NHE3 and NKAα1a were highly conserved among species; NHE3 was mainly involved in low-salt adaptation, while NKAα1a played a role in both low-salt and high-salt adaptation; miR-1335-3p and miR-1788-3p could negatively regulate their target genes NHE3 and NKAα1a, respectively, and thus participate in osmotic pressure regulation in R. canadum. The above results provided a theoretical basis for the further studies of the miRNA-mRNA osmotic pressure regulatory network in R. canadum.