Chondroitin polymerization was first demonstrated in vitro when human chondroitin synthase (ChSy) was coexpressed with human chondroitin polymerizing factor (ChPF), which is homologous to ChSy but has little glycosyltransferase activity. To analyze the biological function of chondroitin, the Caenorhabditis elegans ortholog of human ChSy (sqv-5) was recently cloned, and the expression of its product was depleted by RNA-mediated interference (RNAi) and deletion mutagenesis. Blocking of chondroitin synthesis resulted in defects of cytokinesis in early embryogenesis, and eventually, cell division stopped. Here, we cloned the ortholog of human ChPF in C. elegans, PAR2.4. Despite little glycosyltransferase activity of the gene product, chondroitin polymerization was demonstrated as in the case of mammals when PAR2.4 was coexpressed with cChSy in vitro. The worm phenotypes including the reversion of cytokinesis, observed after the depletion of PAR2.4 by RNAi, were very similar to the cChSy (sqv-5)-RNAi phenotypes. Thus, PAR2.4 in addition to cChSy is indispensable for the biosynthesis of chondroitin in C. elegans, and the two cooperate to synthesize chondroitin in vivo. The expression of the PAR2.4 protein was observed in seam cells, which can act as neural stem cells in early embryonic lineages. The expression was also detected in vulva and distal tip cells of the growing gonad arms from L3 through to the young adult stage. These findings are consistent with the notion that chondroitin is involved in the organogenesis of the vulva and maturation of the gonad and also indicative of an involvement in distal tip cell migration and neural development.