A knockout (KO) of the porcine α1,3-galactosyltransferase (GGTA1) gene is crucial for controlling the hyperacute rejection after pig-to-human xenotransplantation. Porcine kidney and cardiac xenografts from Gal-KO pigs showed prolonged survival after transplantation into baboons. Unfortunately, knockouts produced by conventional targeting (homologous recombination) are rare events and normally do not lead to biallelic KO. Zinc-finger nucleases (ZFN) have been shown to be much more efficient by inducing mutations via specific cleavage followed by nonhomologous end joining (NHEJ). Zinc-finger nucleases do not require antibiotic selection. Here, we used designed ZFN to specifically target exon 9 of the GGTA1 gene encoding the catalytic domain of the Gal-transferase. Recently, we generated female pigs with a GGTA1-KO using ZFN (Hauschild et al. 2011 PNAS 108, 12 013–12 017). Here, we investigated whether cells of a male cell line are susceptible to ZFN-mediated genome editing in a comparable manner. Male porcine fetal fibroblasts (3 × 10⁶) were co-transfected with a ZFN-plasmid pair (7.5 μg each) by electroporation at 250 V and 400 μF. One week after transfection, a Cel-I assay revealed a NHEJ rate of 5.7% of all alleles in the cell population. After magnetic bead selection, Gal-expression was analysed by fluorescence-activated cell sorting (FACS) using fluorescein isothiocyanate (FITC)-conjugated isolectin-B4. Ninety-five percent of the cells were free of Gal epitopes, indicating a biallelic KO. These Gal-negative cells served as donor cells in somatic cell nuclear transfer (SCNT). In total, 507 transgenic embryos were transferred into 6 recipient sows. By obtaining live animals by SCNT after transfer of male ZFN-GGTA1-KO embryos, we will have produced female and male ZFN-KO pigs, which can be used for further breeding experiments to circumvent the extensive and relative inefficient recloning method. These results show that ZFN work independent of the sex of the cells and that a biallelic Gal-KO can be produced in male cells by using the ZFN technology. This technology could benefit both agriculture and biomedicine and establishes the pig as a model for human diseases.