We investigated the reproduction of the amphioxus Branchiostoma belcheri in the southern Ariake Sea, Japan, from 1999 to 2002. Gonads were very small or almost absent from September to December and began to develop in January. Changes in the gonad length index indicated that spawning began in mid June, after the gonads had attained maximum size. Although most gametes were extruded during the first spawning, some remained in the gonads. Shrunken gonads were much smaller but contained gametes, and the second spawning occurred around 10 July. It was unclear whether all adults spawned twice, but a large proportion of individuals did. After the first spawning, both males and females lost 30% of their body weight. Most one-year-old individuals did not spawn, and the minimum size at maturity was ca. 20 mm. We found no termination of reproduction among very large individuals. The average number of gonads was significantly larger on the right side of the body (26.2 in males and 26.1 in females) than on the left side (24.0 in males and 23.5 in females). We found two hermaphrodites in a total of 11,184 specimens examined. Each had four or three ovaries among a total of 45 or 54 gonads.