Gene targeting in livestock fibroblasts has proven extremely difficult to achieve, particularly on silent gene locus. To obtain IgH functional disruption goats used for humanized antibody research, the goat IgH gene, which is transcriptionally silent in fibroblast, was knocked out and the targeted fibroblasts can be used to produce goats with IgH disruption through somatic cell clone. The goat immunoglobulin heavy chain J-Cμ fragment was amplified from the genomic DNA of goat fetal fibroblasts GEF88 through PCR and used as homologous arm to construct an isogenic Positive-Negative Selective targeting vector GTIgH. Then the GEF88 were transfected with the linearized targeting vector GTIgH through electroporation and selected in cell culture medium with 0.8 mg/L puromycin. 1 of the 362 drug-resistant clones was positive for targeting events through PCR screen, and the targeted clone was further confirmed by sequencing and Southern blotting. This suggests that one allele of IgH gene has been successfully knocked out in goat fetal fibroblasts.