Lactococcus lactis ssp. cremoris and Lactobacillus acidophilus NCFM were cultured in growth media supplemented with CaCl2 to induce intracellular accumulation of Ca2+. Both strains grown with 10 mM Ca2+ demonstrated enhanced thermal stability, with increased survival after spray drying from 49.9% to 64.9% for the coccus and from 35.5% to 43.3% for the rod. The increased intracellular Ca2+ level was confirmed with murexide assay. Adding Ca2+ in the reconstitution solution could not improve the viability of spray dried cells at extended storage stages. We hypothesized that the over-presence of intracellular Ca2+ might stimulate the activity of heat shock proteins, increasing cell survival towards heat stress. The positive effect of calcium partly explained the protective mechanism of some calcium-containing protectants. The reported approach, which improves the stress tolerance of cells, can be combined with the optimization of spray drying conditions and protectant formulation for developing a consolidated powder production scheme.