Fishes adapt to salinity changes primarily through osmotic pressure regulation, a process often associated with several genes, including 14-3-3a, NKCCla, APO-14, and Na+-K+-ATPaseβ. The present study investigated the differential expression of genes 14-3-3a, NKCCla, APO-14, and Na+-K+-ATPaseβ in the gill tissue of Mugil cephalus acclimated to low salinity. Susceptibility relationships between the four gene expressions levels and salinity were detected and analyzed using polymerase chain reaction-restriction fragment length polymorphism. Homology analysis results indicated significant differences in the correlation between gene expression and salinity. Under low-salt conditions, expression levels for genes Na+-K+-ATPaseβ and NKCC1a were significantly elevated (P < 0.05), whereas those of genes 14-3-3a and APO-14 were significantly reduced (P < 0.05). Thus, when compared to 14-3-3a and APO-14, Na+-K+-ATPaseβ, and NKCC1a may be better suited to promoting the development of osmotic-regulation mechanisms and increased resistance to environmental stress under low-salt conditions. Furthermore, Na+-K+-ATPaseβ and NKCC1a were identified as suitable potential molecular biomarkers for regulating and controlling genes in low-salinity aquatic environments.