As a key regulator of the cellular balance of pro-apoptotic and pro-survival sphingolipids, the dysregulation of the cell signalling enzyme sphingosine kinase 1 (SK1) is important for various pathological conditions, including cancer. Oncogenic signalling of SK1 relies on its localisation to the plasma membrane (PM), which we have previously shown to be mediated by the calcium and integrin binding (CIB)1 protein . A family of four CIB proteins exist, named CIB1-4, but to date the roles of CIB2-4 have only been poorly defined. Here we show that CIB2 plays a surprisingly opposite role to CIB1 in the regulation of SK1 signalling. CIB2 interacts with SK1 at the same site as CIB1, but unlike CIB1, this interaction occurs in a metal ion-independent manner. Furthermore, despite being myristoylated, CIB2 does not exhibit calcium-myristoyl switch behaviour, which is the mechanism whereby CIB1 mediates PM localisation of SK1. Notably, overexpression of CIB2 inhibited agonist-induced translocation of SK1 to the PM, and consistent with this, blocked cell survival signalling by tumor necrosis factor (TNF)α, and reduced Ras-induced neoplastic transformation of cells, processes that have previously been shown to be dependent on SK1. Together, these results suggest that CIB2 is an endogenous inhibitor of SK1 translocation and its subsequent oncogenic signalling. Further examination of CIB2 expression in a panel of human cancer tissues showed significantly reduced CIB2 mRNA levels in ovarian cancer, compared to normal ovarian tissue. Correspondingly, in the OV3 ovarian cancer cell line, which was found to express very low levels of endogenous CIB2, overexpression of CIB2 resulted in reduced cell migration, similar to that previously observed with SK1 knock-down in these cells. Furthermore, the overexpression of CIB2 also resulted in reduced anchorage-independent growth of the SKOV3 ovarian cancer cells. These studies suggest a potential tumor suppressor role of CIB2 protein in ovarian cancer.