The homeodomain-interacting protein kinase (HIPK) family is comprised of four highly related serine/threonine kinases originally identified as co-repressors for various homeodomain-containing transcription factors. The HIPKs have been shown to be involved in growth regulation and apoptosis, with numerous studies highlighting HIPK regulation of the tumor suppressor p53. In this study, we have discovered a B cell homeostatic defect in HIPK1-deficient (HIPK1(-/-)) mice. Lymphopoietic populations within the thymus and bone marrow of HIPK1(-/-) mice appeared normal based upon FACS analysis; however, the spleen exhibited a reduced number of total B cells with a significant loss of transitional-1 and follicular B cell populations. Interestingly, the marginal zone B cell population was expanded in HIPK1(-/-) mice, yielding an increased frequency of these cells. HIPK1(-/-) B cells exhibited impaired cell division in response to B cell receptor cross-linking in vitro based upon thymidine incorporation or CFSE dilution; however, the addition of CD40L rescued HIPK1(-/-) proliferation to wild-type levels. Despite the expanded MZ B cell population in the HIPK1(-/-) mice, the T-independent type 2 humoral response was impaired. These data identify HIPK1 as a novel kinase required for optimal B cell function in mice.