Na(+)/K(+)-ATPase (NKA) is a widely found and important transporter in mammals. The kidney is a major osmoregulatory organ of which the proximal tubules play a crucial role in the maintenance of ionic homeostasis functioning via salt and water reabsorption. FXYD (FXYD domain-containing protein) 2, the γ-subunit of NKA, is the first identified and the most abundant member of FXYD family, affecting the sodium/potassium affinity of NKA in the kidney. Based on DNA microarray analysis, the expression levels of fxyd2 gene are markedly increased upon hypertonic challenge. Combined with bioinformatic analysis using the NCBI database, we identified an unnamed protein with 145 amino acids, of which the N-terminus involved the FXYD sequence similar to FXYD2a and FXYD2b, and thus, named as FXYD2c. However, the role of FXYD2c protein in the regulation of NKA expression in the kidney has not been elucidated. In this study, we found that the mRNA and protein levels of FXYD2c were significantly increased upon hypertonic challenge. Immunoprecipitation data revealed that FXYD2c interacts with the NKA α1 subunit. Subsequently, the functional inhibition of fxyd2c using short hairpin RNA abrogated NKA activity. Taken together, our study offers novel insight into the potential function of FXYD2c in promoting NKA activity upon hypertonic challenge in HK-2 cells.