The C3 halophyte Suaeda salsa was used to investigate the roles of Ca2+, Ca2+ channels, and calmodulin (CaM) in betacyanin metabolism. Seeds of S. salsa were cultured in both the dark and light for 3 days. The fresh weight and betacyanin content were much higher in S. salsa seedlings formed in the dark than in seedlings formed in the light. The addition of Ca2+ to the half-strength MS nutrient solution promoted betacyanin accumulation in the dark, whereas Ca2+ depletion by EGTA suppressed the dark-induced betacyanin accumulation in shoots of S. salsa. The Ca2+ channel blocker LaCl3 also inhibited dark-induced betacyanin accumulation. The highest activity of CaM and the maximum betacyanin content decreased by 51% and 45%, respectively, in shoots of S. salsa seedlings treated with the potent CaM antagonist chlorpromazine in the dark. Furthermore, the other CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) also inhibited the activity of CaM and dark-dependent betacyanin accumulation, whereas its less active structural analog N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5) had little effect on the responses to dark of S. salsa seedlings. These results suggest that Ca2+, Ca2+-regulated ion channels, and CaM play an important role in dark-induced betacyanin accumulation in the shoots of the C3 halophyte S. salsa.