Cre recombinase (Cre)‐mediated targeted insertion of a transgene is a powerful technique that can be used to tailor genomes. When combined with somatic cell nuclear transfer it could offer an efficient way to generate transgenic livestock with site‐specific genetic modifications that are free of antibiotic selection markers. We have engineered primary bovine fibroblasts to contain a chromosomal acceptor site with incompatible loxP/lox2272 sites for Cre‐mediated cassette exchange and show for the first time that Cre‐mediated targeting can be applied in these acceptor cells. Molecular characterization of the resulting cell clones revealed Cre‐mediated transgene insertion efficiencies of up to 98% when antibiotic selection was used to identify transgene containing cell clones. Most clonal lines also contained random insertions of the targeting and Cre expression plasmids with only about 10% of the clones being exclusively modified by the intended targeted insertion. This targeting efficiency was sufficient to enable the isolation of correctly targeted clones without the help of antibiotic selection. Therefore, this recombinase‐mediated insertion strategy has the potential to produce transgenic cattle from antibiotic selection marker‐free somatic cells with transgenes inserted into proven genomic loci ensuring reliable expression levels.