Foot-and-mouth disease virus (FMDV) is a pathogen of cloven-hoofed livestock with a significant financial burden across the globe. The cost of the 2001 outbreak in the United Kingdom was >£15 billion and there are currently no effective therapies available. Elucidating how FMDV utilises host proteins to support viral replication could present opportunities for the development of therapeutics. FMDV is thought to replicate in membrane-associated replication complexes that contain the viral and host factors essential for replication, however, the precise components of the replication machinery are undescribed. Here, we focus on the essential viral protein 2C. This protein is believed to form hexamers and have RNA binding, chaperone and helicase activity. Using a novel 2C antibody in co-immunoprecipitation experiments under different conditions followed by mass spectrometry, we previously identified membrane-associated proteins along with ER-associated host proteins. In addition, our analysis shows that a number of nuclear factors interact with 2C in a membrane-independent manner.  To identify which host proteins are being co-opted to support viral RNA replication, ten of the most significantly abundant proteins were selected for further investigation. Using siRNA, we demonstrated that one of these proteins, ATP6AP1 (a V-ATPase accessory protein) is important for FMDV RNA replication. Baflomycin A1, a specific inhibitor of V-ATPase, was used to determine if the effect of ATP6AP1 protein knockdown was dependent on its function in the V-ATPase complex. When compared with ATP6AP1 siRNA, Baflomycin A reduced FMDV RNA replication to comparable levels. These data suggest that the V-ATPase is involved in genome replication.