Bone Morphogenetic Proteins (BMPs) plays a pivotal role in dorsoventral (DV) patterning during early embryogenesis. Myristoylated alanine-rich C kinase substrate (MARCKS), a major substrate for protein kinase C, has been shown to regulate cell motility and membrane trafficking. Here, we show the essential role of Marcksb in zebrafish DV patterning. There are 4 MARCKS family genes, marcksa, marcksb, marcksl1a and marcksl1b in zebrafish genome, and marcksb is the only one showing maternal expression. Knockdown of marcksb but not the other 3 MARCKS family members resulted in dorsalization, and the dorsalization defects could be partially rescued by overexpression of each of the 4 genes. The overall BMP signaling activity was strongly reduced in the marcksb morphants, as revealed by analysis of BMP targets and phosphorylated Smad1/5/8 levels. Furthermore, Bmp2b-induced ventralization was blocked by knockdown of marcksb, indicating that Marcksb is required for proper BMP signaling. Mechanically, we found that the mCherry-fused Bmp2b proteins were largely reduced at ex-cellular space in marcksb morphants comparing to wildtype embryos, suggesting that the proper secretion of Bmp2b was disrupted in marcksb morphants. We generated maternal-zygotic mutants of marcksb (MZmarcksb) using TALEN technology. Although marcksb is transcriptionally silent in MZmarcksb, defects of DV patterning was not observed in those embryos. In contrast to the marcksb morphants, the MZmarcksb embryos showed strong upregulation of marcksa, marcksl1a and marcksl1b. This suggests that genetic compensation effects occurred when marcksb was genetically absent. Therefore, our findings reveal a novel function of marcksb in DV patterning possibly by regulating the secretion of BMP ligands.