["Journal of Cellular Physiology, Volume 241, Issue 5, May 2026. ", "\nABSTRACT\nIntracellular calcium (Ca2+) signaling controls myoblast proliferation, fusion, and myofiber formation. In myoblasts, Transient Receptor Potential Canonical (TRPC) channels, with TRPC1 as a predominant isoform, mediate store‐operated Ca²⁺ entry (SOCE) and are essential for myogenesis. PDLIM5 (ENH1), a PDZ‐LIM scaffold protein, organizes signaling events, including ion channel regulation and transcriptional control in muscles. This study aims to test the hypothesis that PDLIM5 regulates TRPC1‐mediated Ca2+ entry in myoblasts. Thapsigargin‐induced SOCE was suppressed by the SOCE inhibitors Gd3+ and 2‐APB, as well as by TRPC1 siRNA, supporting the involvement of TRPC1 in SOCE in C2C12 myoblasts. Additionally, SOCE inhibition decreased the number of nuclei per myotube and reduced the size of myotubes. ENH1 siRNA knockdown significantly downregulated TRPC1 and STIM1 mRNA expression, increased basal cytosolic Ca2+ level, and impaired SOCE response and myotube maturation. Overexpression of ENH4, a skeletal muscle‐specific short splice variant of ENH1, similarly repressed TRPC1‐mediated SOCE and myotube formation. Conversely, ENH1 overexpression enhanced SOCE without altering the mRNA levels of TRPC1, Orai1, or STIM1. Immunoprecipitation showed a physical interaction between ENH1/ENH4 and TRPC1. In differentiated myotubes, TRPC1 also contributed to thapsigargin‐induced SOCE, as evidenced by the reduced Ca2+ entry following TRPC1 knockdown. ENH1 knockdown and ENH4 overexpression significantly attenuated SOCE in myotubes; notably, ENH1 knockdown also increased basal cytosolic Ca2+ level. In contrast to myoblasts, ENH1 overexpression did not enhance SOCE in myotubes, concomitant with the absence of a detectable interaction between ENH1 and TRPC1, whereas ENH4 retained its association with TRPC1. These findings suggest that ENH1 and ENH4 differentially modulate TRPC1‐dependent Ca2+ entry in C2C12 cells, thereby regulating myogenic differentiation and contributing to skeletal muscle formation and development.\n"]