The metabolic network is composed of enzymatic reactions (ERs) in which one or more enzymes catalyze the reaction of pertinent substrates. Since metabolism is a basal system for maintaining life of all organisms, any change in the metabolic networks must greatly affect organismic evolution. The aim of this study is to examine how often gains and losses of ER have occurred during the evolution of metabolic networks in eukaryotes and how these evolutionary events have affected phenotypic traits of organisms. In this study, we conducted comparative studies of 751 ERs in the metabolic networks of 6 eukaryotic species whose complete genome sequences were determined. As a result, we found that a total of 804 gains and losses of ERs had occurred in the evolutionary diversification of metabolic networks in different lineages. Moreover, the vertebrate lineage, after the separation from Drosophila melanogaster, showed a remarkable increase in the number of ER gains compared with ER losses. In particular, 41% of the ER gains were predominantly involved with lipids and complex lipid metabolism. Because some products of these two metabolisms function as hormones, we concluded that the ER gain of these two metabolisms accelerated the development of hormonal signal transduction for the elaborate regulation of physiological systems during vertebrate evolution.