AbstractCalcium intake inhibits growth of colon cancer in vivo, the mechanisms of which are not fully elucidated. The objective of this study was to determine whether Ca2+directly affects the growth of colon cancer cells in vitro and to compare the effects of Ca2+on the growth of several gastroenteropancreatic cancer cells, including mouse colon cancer (MC‐26), human colon cancer (Lo Vo and WIDR), human gastric cancer (AGS and SII), and human pancreatic cancer (PANC‐1 and MIA) cells. All tumor cell lines tested grew in medium containing low concentration (approx 0.16 mM) of Ca2+. Higher concentrations of Ca2+significantly inhibited the growth of all three colon cancer cell lines tested but had no significant effect on proliferation of the stomach and pancreatic cancer cell lines. Growth of AGS cells, in the presence of 0.1 or 0.5 mMEGTA (resulting in the loss of the extracellular Ca2+) was similar to that observed in the absence of EGTA, indicating that AGS cells were relatively insensitive to loss of extracellular Co2+. In the presence of TMB‐8, an inhibitor of intracellular Ca2+release, the growth of colonic cancer cell lines was inhibited in a dose‐dependent manner, indicating that a minimum basal level of intracellular Ca2+was required for continued proliferation of colon cancer cells. The stomach cancer cell lines (AGS) was once again less sensitive to the effects of TMB‐8 than were the colon cancer cells, indicating an inherent difference in Ca2+requirements and sensitivity to Ca2+for growth of different gastroenteropancreatic cancer cells in vitro. These results further demonstrate that in contrast to normal cells, all tumor cells tested can continue to proliferate in the presence of low concentrations of extracellular Ca2+(approx<0.16 mM) and that higher concentrations of Ca2+specifically and directly inhibit growth of human and mouse colon cancer cells. The latter results may provide an additional explanation for the mechanism by which intake of dietary calcium decreases the risk of developing colon cancers.