In vertebrates, glycine amidinotransferase (AGAT), guanidinoacetate methyltransferase (GAMT), and creatine transporter (CT1) are three proteins involved in creatine synthesis. To provide insight into the molecular evolution mechanism of creatine metabolism, we have cloned and identified BbAGAT, BbGAMT, and BbCT1 homologous genes in amphioxus (Branchiostoma belcheri), whose predicted proteins show high identities with AGAT, GAMT, and CT1 proteins in vertebrates. The phylogenetic analysis indicates that amphioxus AGAT, GAMT, and CT1 are branched off at the base of the vertebrate homologous clade, respectively. Genomic structures of BfAGAT, BfGAMT, and BfCT1 show their comparability with the homologs in vertebrate and original characteristic of cephalochordate, which is consistent with animal classification. To determine the expression patterns of BbAGAT, BbGAMT, and BbCT1, whole-mount and section in situ hybridizations are carried out in embryos and adults of amphioxus. During embryogenesis, they are all expressed mainly in mesendoderm and late somites, but BbCT1 is also expressed in differentiating notochord and digestive tract, as well as in the cytoplasm of zygotes and the blastomeres at cleavage stage. In adult, the transcripts of BbAGAT and BbGAMT are detected in the neural cord, gill, nephridia, endostyle, gut, and gonads, while BbCT1 is expressed mainly in the epithelium of gut. The expression pattern of these three genes is similar to their vertebrate homologs. The result reveals that AGAT, GAMT, and CT1, the primary elements of vertebrate creatine metabolism, exist in cephalochordate amphioxus, and are highly conserved during evolution. It also suggests that similar mechanism of creatine synthesis in vertebrate may occur in amphioxus.