Vacuolar ATPases help in maintaining the pH of the vacuoles and thereby play a crucial role in the functioning of vacuolar sodium-proton antiporter. Though the various subunits that make V(1) and V(0) sector have been reported in plants their regulation is not understood completely. We have cloned three different isoforms of vacuolar ATPase subunit c (VHA-c) from Pennisetum glaucum with homologies among themselves varying from 38% to approximately 73% at the nucleic acid level. Using real-time PCR approach we have shown that the three isoforms are regulated in a tissue-specific manner under salinity stress. While isoform III is constitutively expressed in roots and shoots and does not respond to stress, isoform I is upregulated under stress. Isoform II is expressed mainly in roots; however, under salinity stress its expression is downregulated in roots and upregulated in shoots. Tissue specific expression under salinity stress of isoform II was also seen after exogenous application of calcium. This study for the first time shows the presence of three isoforms of PgVHA-c and their differential regulation during plant development, and also under abiotic stress.