We describe the development of a new transformation system, using multiple auxotrophic marker genes, for the fission yeast Schizosaccharomyces pombe. We developed three new auxotrophic marker genes (arg12(+), tyr1(+) and ade7(+)) and generated a new host strain, YF043, by Cre-loxP-mediated gene disruption. YF043 possessed six mutated biosynthetic genes (leu1-32, ura4-M190T, arg12::loxP, tyr1::loxP, ade7::loxP and his2::loxP). The combination of this host strain and the new selectable markers can be used for gene disruption using the same preexisting transformation systems. In addition, Sz. pombe vectors were constructed, containing selectable marker genes that complement the auxotrophies of YF043. These new vectors are available for gene disruption and heterologous protein expression in strain YF043. The new Sz. pombe host strain will be a useful tool for molecular genetic studies of Sz. pombe where multiple recombinant modifications or multiple mutations are needed.