Foot-and-mouth disease virus (FMDV) remains an ever-present threat to the economic stability of the livestock industry and global trade. Despite this, questions remain regarding the fundamental biology underpinning the replication of this virus. Here, we examine components of the FMDV replication machinery (focussing on the viral 2C protein) and investigate the conditions under which they interact. Using a novel 2C antibody in co-immunoprecipitation experiments under different conditions followed by MS, we identify membrane-associated proteins (such as the viral proteins 2B and precursors of 3A, which are poorly characterized proteins involved in viral replication) along with endoplasmic reticulum (ER)-associated host proteins. In addition, our analysis shows that a number of nuclear factors interact with 2C in a membrane-independent manner, potentially being co-opted to support RNA replication of the virus. Furthermore, we demonstrate that the membrane-mediated interaction of 2C with several viral proteins (including key members of the replication machinery and viral RNA) is maintained following ultracentrifugation, suggesting that these co-sediment as part of a complex. Our data suggest that the replication complex is ER-derived and highlight several new avenues of investigation for the disruption of the FMDV lifecycle.