Technical Abstract: Foot-and-mouth disease virus (FMDV), the causative agent of foot-and-mouth disease (FMD), is an Apthovirus within the Picornaviridae family. Replication of the virus occurs in association with replication complexes that are formed by host cell membrane rearrangements. The largest viral protein in the replication complex, 2C, is thought to have multiple roles during virus replication. However, studies examining the function of FMDV 2C have been rather limited. To better understand the role of 2C in the process of virus replication we used a yeast two-hybrid approach to identify host proteins that interact with 2C. We report here that cellular Beclin1 is a specific host binding partner for 2C. Beclin1 is a regulator of the autophagy pathway, a metabolic pathway required for efficient FMDV replication. The 2C-Beclin1 interaction initially identified by the yeast two-hybrid approach was further confirmed by co-immunoprecipitation and confocal microscopy to actually occur in FMDV infected cells. Over-expression of either Beclin1 or Bcl-2, another important autophagy factor, strongly affects virus yield in cell culture. The fusion of lysosomes to autophagosomes containing viral proteins is not seen during FMDV infection, a process that is stimulated by Beclin1; however, in FMDV-infected cells over-expressing Beclin1 this fusion occurs, suggesting that 2C binds to Beclin1 to prevent the fusion of lysosomes to autophagosomes, allowing for virus survival. The critical amino acid residues in 2C that mediate the interaction with Beclin1 were mapped in detail using alanine scanning mutagenesis. Using reverse genetics we demonstrated that modifications to 2C in areas critical for interaction with Beclin1 are also critical for virus growth. These results suggest that interaction between FMDV 2C and host protein Beclin1 could be essential for virus replication.