Nervonic acid (24:1) is a major component in nerve and brain tissues and it has important applications in food and pharmaceutical industries. Malania oleifera seeds contain about 40% nervonic acid. However, the mechanism of nervonic acid biosynthesis and accumulation in seeds of this endangered tree species remains unknown. In this study, developmental changes in fatty acid composition within embryos and their pericarps were investigated. Nervonic acid proportions steadily increased in developing embryos but 24:1 was not detected in pericarps at any stage. Two 3-ketoacyl-CoA synthase (KCS) homologs have been isolated from M. oleifera developing seeds by homologous cloning methods. Both KCSs are expressed in developing embryos but not detected in pericarps. Based on a phylogenetic analysis, these two KCSs were named as MoKCS4 and MoKCS11. Seed-specific expression of the MoKCS11 in Arabidopsis thaliana led to about 5% nervonic acid accumulation, while expression of the MoKCS4 did not show an obvious change in fatty acid composition. It is noteworthy that the transformation of the same MoKCS11 construct into two Brassica napus cultivars with high erucic acid did not produce the expected accumulation of nervonic acid, although expression of MoKCS11 was detected in the developing embryos of transgenic lines. In contrast, overexpression of MoKCS11 results in similar level of nervonic acid accumulation in camelina, a species which contains a similar level of 11Z-eicosenoic acid as does Arabidopsis thaliana. Taken together, the MoKCS11 may have a substrate preference for 11Z-eicosenoic acid, but not for erucic acid, in planta.