Intracellular Ca2+ fluxes regulate fundamental cellular processes such as proliferation, differentiation and death. Not surprisingly, an alteration of the mechanisms that govern these fluxes results in disease. Cancer cells, which are characterised by enhanced proliferation and insensitivity to death-inducing stimuli, may acquire these characteristics by remodelling their Ca2+ signalling toolkit. In support of this, recent studies report crosstalk between Ca2+ and oncogenic signalling pathways; moreover, mutations or altered expression of Ca2+-handling proteins occurs frequently in cancer. It is however still unclear how this remodelling takes place and whether it is a cause or consequence of cancer onset. In this study we investigated the hypothesis that altered Ca2+ signals underlie the phenotypic changes associated with oncogenic transformation. To this end, we characterised Ca2+ signalling in a human colorectal cancer model (the K-Ras mutated cell line HCT116) and in the appropriate control HKH2 (which was obtained by targeted deletion of the mutated allele)[1]. Experiments were also validated on the colorectal cancer cell line DLD-1 paired with the isogenic derivative cell line DKO4. Single cell calcium imaging experiments revealed diminished ATP-stimulated calcium signals in HCT116 compared to HKH2 (in all experiments: numbers±S.E.M., n=12 coverslips with >60 cells each, significance in Student t-test; for 2 uM ATP, integrated response (AUC): HCT116 4547±2396, HKH2 13813±2544; % of responders (%resp): HCT116 0.38±0.32, HKH2 0.87±0.13). This effect was conserved in the absence of extracellular Ca2+ (AUC: HCT116 8740±742.7, HKH2 12630±884.0; number of responders (no.resp): HCT116 48±7, HKH2 78±6) and when Ca2+ was released from intracellular stores with membrane-permeant InsP3 (AUC: HCT116 16040±2974, HKH2 32800±4903; no.resp: HCT116 42±6, HKH2 90.5±4). Whether the decreased calcium release was due to a reduction in endoplasmic reticulum (ER) store content or InsP3 receptor expression was therefore tested. Administration of the sarco-endoplasmatic reticulum Ca2+ ATP-ase (SERCA) blocker thapsigargin resulted in reduced calcium release in HCT116 cells (AUC: HCT116 106106±8494, HKH2 140696±7733). Moreover, expression of IP3Rs was modified in HCT116 cells compared to HKH2, whereby type I IP3R was increased and type III IP3R decreased (n=8 replicates). These data suggest that the decreased IP3-stimulated Ca2+ release observed in HCT116 cells is due to both a reduction in ER store content together with a remodelling of IP3R isoform expression. These modifications in Ca2+ homeostatic mechanisms may serve to provide a survival advantage during oncogenic transformation. We anticipate that this study will increase our understanding of the complex relationship between calcium and cancer signalling, and may identify new targets for the development of therapy.