Loss of the major isoform of phosphoglucomutase (PGM) causes an accumulation of glucose 1-phosphate when yeast cells are grown with galactose as the carbon and energy source. Remarkably, the pgm2Δ strain also exhibits a severe imbalance in intracellular Ca2+ homeostasis when grown under these conditions. In the present study, we examined how the pgm2Δ mutation alters yeast Ca2+ homeostasis in greater detail. We found that a shift from glucose to galactose as the carbon source resulted in a 2-fold increase in the rate of cellular Ca2+ uptake in wild-type cells, whereas Ca2+ uptake increased 8-fold in the pgm2Δ mutant. Disruption of the PMC1 gene, which encodes the vacuolar Ca2+-ATPaSe Pmc1p, suppressed the Ca2+-related phenotypes observed in the pgm2Δ strain. This suggests that excessive vacuolar Ca2+ uptake is tightly coupled to these defects in Ca2+ homeostasis. An in vitro assay designed to measure Ca2+ sequestration into intracellular compartments confirmed that the pgm2Δ mutant contained a higher level of Pmc1p-dependent Ca2+ transport activity than the wild-type strain. We found that this increased rate of vacuolar Ca2+ uptake also coincided with a large induction of the unfolded protein response in the pgm2Δ mutant, suggesting that Ca2+ uptake into the endoplasmic reticulum compartment was reduced. These results indicate that the excessive Ca2+ uptake and accumulation previously shown to be associated with the pgm2Δ mutation are due to a severe imbalance in the distribution of cellular Ca2+ into different intracellular compartments.