Calcium signaling is important for multiple events during embryonic development. However, roles of calcium influx during embryogenesis have not been fully understood since routes of calcium influx are often redundant. To define roles of voltage-gated calcium channel (Cav) during embryogenesis, we have isolated an ascidian Cav beta subunit gene (TuCavbeta) and performed gene knockdown using the morpholino antisense oligonucleotide (MO). The suppression of Cav activity by TuCavbetaMO remarkably perturbed gastrulation and tail elongation. Further, larvae with normal morphology also failed to exhibit motility. Phalloidin-staining showed that arrangement of myofibrils was uncoordinated in muscle cells of TuCavbetaMO-injected larvae with normal tail. To further understand the roles of Cav activity in myofibrillogenesis, we tested pharmacological inhibitions with ryanodine, curare, and N-benzyl-p-toluensulphonamide (BTS). The treatment with ryanodine, an intracellular calcium release blocker, did not significantly affect the motility and establishment of the myofibril orientation. However, treatment with curare, an acetylcholine receptor blocker, and BTS, an actomyosin ATPase specific inhibitor, led to abnormal motility and irregular orientation of myofibrils that was similar to those of TuCavbetaMO-injected larvae. Our results suggest that contractile activation regulated by voltage-dependent calcium influx but not by intracellular calcium release is required for proper arrangement of myofibrils.