Despite advances in the study on animal evolution in the last two decades, paucity of experimental data on cephalochordates comparable to those on the other chordates hinders an integrative understanding of chordate evolutionary history. To obtain lancelet data under well-controlled experiments, laboratory cultures of lancelets have been performed at several institutions. In a mass culture started in 2005, we have obtained up to three consecutive generations of Branchiostoma japonicum. Using sand substratum, survival rates of laboratory lancelets until maturation have improved to greater than 30%, much higher than compared to previously, and for adults the annual average survival rate was 82.3%. The high survival rate allows maintaining animals at least 6 years and potentially longer. Water temperatures lower than 23°C obviously reduced the frequency of spawning even after the onset of spawning period, and 1–2 days after changing the temperature at 25°C animals became spawned well. We also observed obvious sex reversal from male to female in individuals that had been cultured for 3 years or more. Our continuous culture has provided sufficient materials for vital experiments on early development and for studying metamorphosis, as well as for the conservation of wild populations. The subculture of successive laboratory generations will provide a valuable resource for genetic studies. J. Exp. Zool. (Mol. Dev. Evol.) 320B: 538–547, 2013. © 2013 Wiley Periodicals, Inc.