The Rho family of small GTPases control cell migration, cell invasion and cell cycle. Many of these processes are perturbed in cancer and several family members show altered expression in a number of tumor types. RhoBTB2/DBC2 is an atypical member of this family of signaling proteins, containing two BTB domains in addition to its conserved Rho GTPase domain. RhoBTB2 is mutated, deleted or silenced in a large percentage of breast and lung cancers; however, the functional consequences of this loss are unclear. Here we use RNA interference in primary human epithelial cells to mimic the loss of RhoBTB2 seen in cancer cells. Through microarray analysis of global gene expression, we show that loss of RhoBTB2 results in downregulation of CXCL14-a chemokine that controls leukocyte migration and angiogenesis, and whose expression is lost through unknown mechanisms in a wide range of epithelial cancers. Loss of RhoBTB2 expression correlates with loss of CXCL14 secretion by head and neck squamous cell carcinoma cell lines, whereas reintroduction of RhoBTB2 restores CXCL14 secretion. Our studies identify CXCL14 as a gene target of RhoBTB2 and support downregulation of CXCL14 as a functional outcome of RhoBTB2 loss in cancer.