Members of the Enterovirus (poliovirus [PV], coxsackieviruses, and human rhinoviruses) and Kobuvirus (Aichi virus) genera in the Picornaviridae family rely on PI4KIIIbeta (phosphatidylinositol-4-kinase IIIbeta) for efficient replication. The small membrane-anchored enteroviral protein 3A recruits PI4KIIIbeta to replication organelles, yet the underlying mechanism has remained elusive. Recently, it was shown that kobuviruses recruit PI4KIIIbeta through interaction with ACBD3 (acyl coenzyme A [acyl-CoA]-binding protein domain 3), a novel interaction partner of PI4KIIIbeta. Therefore, we investigated a possible role for ACBD3 in recruiting PI4KIIIbeta to enterovirus replication organelles. Although ACBD3 interacted directly with coxsackievirus B3 (CVB3) 3A, its depletion from cells by RNA interference did not affect PI4KIIIbeta recruitment to replication organelles and did not impair CVB3 RNA replication. Enterovirus 3A was previously also proposed to recruit PI4KIIIbeta via GBF1/Arf1, based on the known interaction of 3A with GBF1, an important regulator of secretory pathway transport and a guanine nucleotide exchange factor (GEF) of Arf1. However, our results demonstrate that inhibition of GBF1 or Arf1 either by pharmacological inhibition or depletion with small interfering RNA (siRNA) treatment did not affect the ability of 3A to recruit PI4KIIIbeta. Furthermore, we show that a 3A mutant that no longer binds GBF1 was capable of recruiting PI4KIIIbeta, even in ACBD3-depleted cells. Together, our findings indicate that unlike originally envisaged, coxsackievirus recruits PI4KIIIbeta to replication organelles independently of ACBD3 and GBF1/Arf1. IMPORTANCE: A hallmark of enteroviral infection is the generation of new membranous structures to support viral RNA replication. The functionality of these "replication organelles" depends on the concerted actions of both viral nonstructural proteins and co-opted host factors. It is thus essential to understand how these structures are formed and which cellular components are key players in this process. GBF1/Arf1 and ACBD3 have been proposed to contribute to the recruitment of the essential lipid-modifying enzyme PI4KIIIbeta to enterovirus replication organelles. Here we show that the enterovirus CVB3 recruits PI4KIIIbeta by a mechanism independent of both GBF1/Arf1 and ACBD3. This study shows that the strategy employed by coxsackievirus to recruit PI4KIIIbeta to replication organelles is far more complex than initially anticipated.