Anoctamin 5 (ANO5) is a membrane protein belonging to the TMEM16/Anoctamin family and its deficiency leads to the development of limb girdle muscular dystrophy R12 (LGMDR12). However, little has been known about the interactome of ANO5 and its cellular functions. In this study, we exploited a proximal labeling approach to identify the interacting proteins of ANO5 in C2C12 myoblasts stably expressing ANO5 tagged with BioID2. Mass spectrometry identified 41 unique proteins specifically from ANO5-BioID2 samples but not BioID2 fused with ANO6 or MG53, including BVES and POPDC3. The interaction between ANO5 and BVES was further confirmed by coimmunoprecipitation (Co-IP), and the N-terminus of ANO5 mediated the interaction with BVES through its C-terminus. ANO5 and BVES were co-localized in muscle cells and enriched at the endoplasmic reticulum (ER) membrane. Genome editing-mediated ANO5 or BVES disruption signi¦cantly suppressed C2C12 myoblast differentiation with little impact on proliferation. Taken together, these data suggest that BVES is a novel interacting protein of ANO5, involved in regulation of muscle differentiation.