CTP synthase (CTPS) is a rate-limiting enzyme that controls CTP synthesis and can polymerize to form a filamentous structure called cytoophidia. The presence of cytoophidia affects the enzymatic activity of CTPS. However, whether CTPS can form cytoophidia in C2C12 cells and whether it affects the proliferation of skeletal muscle satellite cells needs to be further studied. In this study, we found that CTPS could form cytoophidia during C2C12 cell proliferation, and that overexpression of CTPS significantly promoted the formation of CTPS cytoophidia and increased the viability and proliferation rate of C2C12 cells. However, the CTPS H355A mutation hindered the formation of CTPS cytoophidia and inhibited the viability and proliferation of C2C12 cells. In addition, we found that the HSPD1 protein could interact with the CTPS protein and interference with Hspd1 gene expression inhibited the formation of CTPS cytoophidia, even with the overexpression of the CTPS gene. Subsequently, it inhibited C2C12 cells proliferation. Thus, these findings reveal the role of CTPS cytoophidia formation in C2C12 cells proliferation.