The Rad51-like proteins, Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3, have been shown to form two distinct complexes and seem to assist Rad51 in the early stages of homologous recombination. Although these proteins share sequence similarity with Rad51, they do not show functional redundancy. Among them, Rad51B is unique in that the gene maps to the human chromosome 14q23-24, the region frequently involved in balanced chromosome translocations in benign tumors particularly in uterine leiomyomas. Despite accumulating descriptive evidence of altered Rad51B function in these tumors, the biological significance of this aberration is still unknown. To assess the significance of reduced Rad51B function, we deleted the gene in the human colon cancer cell line HCT116 by gene targeting. Here, we show that haploinsufficiency of RAD51B causes mild hypersensitivity to DNA-damaging agents, a mild reduction in sister chromatid exchange, impaired Rad51 focus formation, and an increase in chromosome aberrations. Remarkably, haploinsufficiency of RAD51B leads to centrosome fragmentation and aneuploidy. In addition, an approximately 50% reduction in RAD51B mRNA levels by RNA interference also leads to centrosome fragmentation in the human fibrosarcoma cell line HT1080. These findings suggest that the proper biallelic expression of RAD51B is required for the maintenance of chromosome integrity in human cells.