Background: L-type calcium channels (LTCC) regulate calcium entry into cardiomyocytes. CACNB2 (β2) LTCC auxiliary subunits traffic the pore-forming CACNA subunit to the membrane and modulate channel kinetics. β2 is a membrane associated guanylate kinase (MAGUK) protein. A major role of MAGUK proteins is to scaffold cellular junctions and multiprotein complexes. Results: To investigate developmental functions for β2.1, we depleted it in zebrafish using morpholinos. β2.1-depleted embryos developed compromised cardiac function by 48 hr postfertilization, which was ultimately lethal. β2.1 contractility defects were mimicked by pharmacological depression of LTCC, and rescued by LTCC stimulation, suggesting β2.1 phenotypes are at least in part LTCC-dependent. Morphological studies indicated that β2.1 contributes to heart size by regulating the rate of ventricle cell proliferation, and by modulating the transition of outer curvature cells to an elongated cell shape during chamber ballooning. In addition, β2.1-depleted cardiomyocytes failed to accumulate N-cadherin at the membrane, and dissociated easily from neighboring myocytes under stress. Conclusions: Hence, we propose that β2.1 may also function in the heart as a MAGUK scaffolding unit to maintain N-cadherin-based adherens junctions and heart tube integrity. Developmental Dynamics 241:648–662, 2012. © 2012 Wiley Periodicals Inc.