The Cre/loxP system is a strategy for controlling temporal and/or spatial gene expression through genome alteration in mice. As successful Cre/loxP genome alteration depends on Cre-driver mice, Cre-reporter mice are essential for validation of Cre gene expression in vivo. In most Cre-reporter mouse strains, although the presence of reporter product indicates the expression of Cre recombinase, it has remained unclear whether a lack of reporter signal indicates either no Cre recombinase expression or insufficient reporter gene promoter activity. We produced a novel ROSA26 knock-in Cre-reporter C57BL/6N strain exhibiting green emission before and red after Cre-mediated recombination, designated as strain R26GRR. Ubiquitous green fluorescence and no red fluorescence were observed in R26GRR mice. To investigate the activation of tdsRed, EGFP-excised R26GRR, R26RR, mice were produced through the crossing of C57BL/6N mice with R26GRR/Ayu1-Cre F1 mice. R26RR mice showed extraordinarily strong red fluorescence in almost all tissues examined, suggesting ubiquitous activation of the second reporter in all tissues after Cre/loxP recombination. Moreover, endothelial cell lineage and pancreatic islet-specific expression of red fluorescence were detected in R26GRR/Tie2-Cre F1 mice and R26GRR /Ins1-Cre F1 mice, respectively. These results indicated that R26GRR mice are a useful novel Cre-reporter mouse strain. In addition, R26GRR mice with a pure C57BL/6N background represent a valuable source of green-to-red photoconvertible cells following Cre/loxP recombination for application in transplantation studies. The R26GRR mouse strain will be available from RIKEN BioResource Center (http://www.brc.riken.jp/lab/animal/en/).