In skeletal muscle, calmodulin (CaM) regulates excitation-contraction coupling, primarily via modulation of ryanodine receptors. Here we aimed to further our understanding of the role of endogenous CaM in excitation-contraction coupling. Since systemic ablation of CaM in mice is difficult to achieve due to CaM's multiple functions, in vivo gene transfer via electroporation mediated transfection method was used to deliver plasmid coding for both cerulean and short-hairpin (sh)RNA targeting CaM (shRNA-CaM) to study the effect of CaM knockdown in adult mouse flexor digitorum brevis skeletal muscle. CaM protein expression levels were significantly reduced in shRNA-CaM fibers, which exhibited no evident morphological changes when compared to the shRNA-control fibers. After confirming the reduction of endogenous CaM expression, we used high-speed confocal microscopy and rhod2-based Ca2+ imaging to assess the consequence of CaM knowdown on action potential (AP)-evoked Ca2+ signals. Isolated single muscle fibers expressing shRNA-CaM exhibited decreased mean peak amplitude and slowed decaying phase of AP-induced Ca2+ transient when compared to the shRNA-controls, indicating compromised Ca2+ release and Ca2+ uptake. We also used a model for myoplasmic Ca2+ binding and transport processes to calculate AP-evoked sarcoplasmic reticulum Ca2+ release flux, which demonstrated decreased Ca2+ release flux and indicated suppressed Ca2+ uptake in shRNA-CaM fibers. Decreased Ca2+ release could reflect decreased coupling between Cav1.1 and ryanodine receptor, a reduction in expression of one or both proteins or a decreased store content, whereas a slowed decaying phase is consistent with compromised Ca2+ uptake. Our study shows the importance of endogenous CaM in the maintenance of excitation-contraction coupling in adult skeletal muscle and could provide new avenues to further explore the potential role of both CaM-dependent and CaM-independent pathways in skeletal muscle contractility and plasticity. Supported by NIH-NIAMS Grant R37-AR055099.