Immunoglobulin V-region somatic hypermutation and C-region class-switch recombination are initiated by activation-induced cytidine deaminase (AID) in B-cells. AID-induced DNA damage at the immunoglobulin S-region is known to be repaired by non-homologous end-joining, but repair mechanisms at the V-region remain to be elucidated. In Saccharomyces cerevisiae, DNA homologous recombination is regulated by the expression of Sac3, involved in actin assembly, cell cycle transition and mRNA metabolism. Here, we demonstrate that the Sac3-homologue GANP suppresses DNA recombination in a direct-repeat beta-galactosidase gene construct in mammalian cells. Homozygous ganp gene knockout is embryonic lethal in mice. Embryonic fibroblasts immortalized from hetero-deficient ganp(+/-) mice showed more DNA recombination than wild-type. In contrast, over-expression of GANP suppressed either spontaneous DNA recombination or that caused by the introduction of aid cDNA into NIH3T3 cells (susceptible to I-sceI restriction enzyme cleavage but not to RAG-mediated immunoglobulin gene recombination). GANP suppresses the DNA recombination not only on the extrachromosomal DNA construct but also on the integrated DNA. The Sac3-homology portion is necessary for the suppressive activity, but the truncated carboxyl terminal MCM3-binding/acetylating region adversely augmented DNA recombination, acting as a dominant negative form. Expression of full-length GANP is critical for suppression of DNA hyper-recombination in mammalian cells.