Blossom-end rot (BER) in tomato fruit (Solanum lycopersicum) is believed to be a calcium (Ca2+) deficiency disorder, but the mechanisms involved in its development are poorly understood. Our hypothesis is that high expression of pectin methylesterases (PMEs) increases Ca2+ bound to the cell wall, subsequently decreasing Ca2+ available for other cellular functions and thereby increasing fruit susceptibility to BER. The objectives of this study were to evaluate the effect of PME expression, and amount of esterified pectins and Ca2+ bound to the cell wall on BER development in tomato fruit. Wild-type and PME-silenced tomato plants were grown in a greenhouse. At full bloom, flowers were pollinated and Ca2+ was no longer provided to the plants to induce BER. Our results show that suppressing expression of PMEs in tomato fruit reduced the amount of Ca2+ bound to the cell wall, and also reduced fruit susceptibility to BER. Both the wild-type and PME-silenced fruit had similar total tissue, cytosolic and vacuolar Ca2+ concentrations, but wild-type fruit had lower water-soluble apoplastic Ca2+ content and higher membrane leakage, one of the first symptoms of BER. Our results suggest that apoplastic water-soluble Ca2+ concentration influences fruit susceptibility to Ca2+ deficiency disorders.