In order to study the function V-ATPase H gene expression on salinity stress in Sinonovacula constricta, the adults were used as experimental materials, and the stress was tested in different salinities (5, 15, 20, 25, 35). The freezing point osmometer was used to determine the difference in serum osmotic pressure, and V-ATPase ELISA detection kit was used to determine the activity of V-ATPase. The full-length ORF sequence of V-ATPase H gene was closed and its mRNA expression was analyzed. The results showed that the serum osmolarity of the low-salt group (salinity 5, salinity 15) and that of high-salt group (salinity 25, salinity 35) changed significantly, both were very significantly different from the control group (salinity 20) (P < 0.01). With the treatment time, the V-ATPase activity of the experimental group showed a decreasing first and then increasing trend, while the control group showed no significant change. The open reading frame (ORF) of the V-ATPase H gene was 1 440 bp in length, encoding 479 amino acids. The qPCR results showed that the expression level of V-ATPase H gene in the gills of S. constricta was significantly higher than that in the other 6 tissues including siphon, mantle, kidney, digestive gland, flabs, and foot (P < 0.01). Under salinity stress, the expression of V-ATPase H gene in the gills of each experimental group continued to rise, peaking at 24 h, which was significantly higher than that of the control group (P < 0.05). The experimental results show that the V-ATPase H gene plays a role in maintaining the balance between its own serum osmotic pressure and the external osmotic pressure in low-salt and high-salt environments during salinity adaptation of S. constricta.