The yolk syncytial layer (YSL) in the zebrafish embryo is a multinucleated syncytium essential for embryo development, but the molecular mechanisms underlying YSL formation remain largely unknown. Here we show that zebrafish solute carrier family 3 member 2 (Slc3a2) is expressed specifically in the YSL and that slc3a2 knockdown causes severe YSL defects including clustering of the yolk syncytial nuclei and enhanced cell fusion, accompanied by disruption of microtubule networks. Expression of a constitutively active RhoA mimics the YSL phenotypes caused by slc3a2 knockdown, whereas attenuation of RhoA or ROCK activity rescues the slc3a2-knockdown phenotypes. Furthermore, slc3a2 knockdown significantly reduces tyrosine phosphorylation of c-Src, and overexpression of a constitutively active Src restores the slc3a2-knockdown phenotypes. Our data demonstrate a signaling pathway regulating YSL formation in which Slc3a2 inhibits the RhoA/ROCK pathway via phosphorylation of c-Src to modulate YSL microtubule dynamics. This work illuminates processes at a very early stage of zebrafish embryogenesis and more generally informs the mechanism of cell dynamics during syncytium formation.