V-ATPase plays a central role in lepidopteran midgut ion transport physiology, and lepidopteran midgut turned out to be a model tissue for the study of V-ATPase. In the present study, the 5'-RACE method is used to obtain the 5'-UTR of V-ATPase c subunit gene from Bombyx mori. Sequence analysis of the promoter region and 3'-UTR of V-ATPase c subunit gene revealed that the transcription of the V-ATPase c subunit gene may be regulated by multi-ways. RT-PCR analysis showed that B. mori V-ATPase c subunit mRNA expresses in the whole developmental stages of B. mori. We also constructed a transient vector to determine the subcellular localization of the B. mori V-ATPase c subunit, and the result demonstrated that it is located in the membrane and some specific regions of BmN cells. Real-time PCR analysis further indicated that the c subunit mRNA expression was upregulated significantly at 24 and 72 h in the midguts of resistant B. mori larvae after being inoculated with B. mori nucleopolyhedrovirus, suggesting that it may be related to the immune response against virus infection.