Although charophycean algae form a relevant monophyly with embryophytes and hence occupy a fundamental place in the development of Streptophyta, no tools for genetic transformation in these organisms have been developed. Here we present the first stable nuclear transformation system for the unicellular Zygnematales, the Closterium peracerosum-strigosum-littorale complex (C. psl complex), which is one of the most useful organisms for experimental research on charophycean algae. When a vector, pSA106, containing the dominant selectable marker ble (phleomycin-resistant) gene and a reporter cgfp (Chlamydomonas-adapted green fluorescent protein) gene was introduced into cells via particle bombardment, a total of 19 phleomycin-resistant cells were obtained in the presence of a low concentration of phleomycin. Six isogenic strains isolated using conditioned medium showed consecutive cgfp expression and long-term stability for phleomycin resistance. DNA analyses verified single or tandem/redundant integration of ~10 copies of pSA106 into the C. psl complex genome. We also constructed an overexpression vector, pSA1102, and then integrated a CpPI gene encoding minus-specific sex pheromone into pSA1102. Ectopic overexpression of CpPI and the pheromonal function were confirmed when the vector pSA1102_CpPI was introduced into mt(+) cells. The present efficient transformation system for the C. psl complex should provide not only a basis for molecular investigation of Closterium but also an insight into important processes in early development and evolution of Streptophyta.