Recently, it has become common to produce transgenic cattle by somatic cell nuclear transfer. As a major step, donor cells are transfected with expression vectors that contain not only the target gene but also marker genes such as neomycin resistant gene (neoR) used for subsequent cell screening. However, such markers in transgenic animals may be undesirable for further researches (Kuroiwa et al. 2004 Nature Genetics 36, 775–780). Therefore, the aim of this study was to excise the marker gene (neoR) in transgenic cattle by using transiently expressed CRE recombinase. Initially, the presence of loxP sites flanking neoR was confirmed in fibroblast cells derived from human lysozyme transgenic cloned cattle. By using cassettes from plasmids PBS185, pIRES-EGFP and pIREShyg3, IRES-EGFP element was inserted downstream of the CRE coding region, and the co-expression vector, which permits translation of GFP and CRE from one messenger RNA, was constructed. Then transgenic fibroblast cells were transfected by the CRE expression vector in circular format by Lipofectamine^™ 2000 and screened by FACS after 48 h. GFP positive cells were selected to culture for 7 to 9 days, and removal of neoR was confirmed by PCR with 2000 to 3000 cells from each colony and the remaining cells used as donors for recloning. Then cleavage and blastocyst rate were evaluated; neoR excision was identified by single blastocyst PCR. Some blastocysts were harvested for differential staining and some used to conduct embryo transfer to evaluate in vitro and in vivo developmental ability. Data were analyzed using SAS (version 9.1) with one-way ANOVA. All experiments were replicated at least three times. We confirmed that loxP sites are correctly located at two sides of neoR. After transfection, neoR was removed from transgenic cloned fibroblast cells and cre was found to be expressed transiently. Additionally, neoR excision was confirmed by single blastocyst PCR. Furthermore, no significant difference was found on cleavage (P = 0.15) and blastocyst rate (P = 0.73) between cloned embryos when using neoR free donors and neoR included donors. The differential staining study showed similar numbers in terms of total cell number (P = 0.69) and the ratio of ICM to total cell number (P = 0.96). After embryo transfer, successful establishment of pregnancies were observed and pregnancy rate on Day 90 and Day 120 (Day 1 is the 1st day after embryo transfer) are similar with control (P > 0.05). In conclusion, we successfully excised resistant marker gene from transgenic cloned cattle by transient expression of CRE recombinase and we expect it will benefit bioreactor and animal transgenic research in the future.