CACNB genes encode membrane-associated guanylate kinase (MAGUK) proteins once thought to function exclusively as auxiliary β subunits in assembly and gating of voltage-gated Ca²⁺ channels. Here, we report that zygotic deficiency of zebrafish β4 protein blocks initiation of epiboly, the first morphogenetic movement of teleost embryos. Reduced β4 function in the yolk syncytial layer (YSL) leads to abnormal division and dispersal of yolk syncytial nuclei, blastoderm retraction, and death, effects highly similar to microtubule disruption by nocodazole. Epiboly is restored by coinjection of human β4 cRNA or, surprisingly, by mutant cRNA encoding β4 subunits incapable of binding to Ca²⁺ channel α1 subunits. This study defines a YSL-driven zygotic mechanism essential for epiboly initiation and reveals a Ca²⁺ channel-independent β4 protein function potentially involving the cytoskeleton.