The cephalochordate, amphioxus, is phylogenetically placed at the most primitive position in the chordate clade. Despite many studies on the endocrine system of amphioxus, definitive evidence has not been reported for the presence an endocrine system comparable to the pituitary-gonadal axis, which is important in the regulation of reproduction in vertebrates. Recent genome analyses in the amphioxus, Branchiostoma floridae, showed that it does not have any pituitary hormone genes except the thyrostimulin gene. Thyrostimulin is a heterodimeric glycoprotein hormone consisting of α and β subunits, and is present in various organs of vertebrates. Analyses of a phylogenetic tree and a synteny suggest that amphioxus' thyrostimulin is an ancestral type of the glycoprotein hormones in chordates. In addition, genes for sex steroidogenic enzymes belonging to the CYP family were found in the genome sequences. The conversion pathway of sex steroids from cholesterol to estrogen, androgen, and major sex steroids was also identified in the gonads of amphioxus in vitro. Furthermore, we demonstrated the expression of genes encoding thyrostimulin and sex steroidogenic enzymes by an in situ hybridization technique. Here, we discuss the evolution of hormones and reproductive functions in the neuroendocrine control system of chordates.