Glycolytic metabolism in meiotic and post-meiotic spermatogenic cells shows differentiation-related changes. The developmental and physiological significance of these metabolic changes is not known. The aim of the present study was to test the hypothesis that glucose and lactate metabolism can modulate intracellular calcium [Ca2+]i in spermatogenic cells in an opposing and dynamic manner. Fluorescent probes were used to measure [Ca2+]i and pHi, and HPLC was used to measure intracellular adenine nucleotides and mitochondrial sensing of ATP turnover. [Ca2+]i in pachytene spermatocytes and round spermatids was modulated by changes in lactate and glucose concentrations in the media. The kinetics and magnitude of the [Ca2+]i changes induced by lactate and glucose were different in meiotic and post-meiotic spermatogenic cells. The presence of glucose in the medium induced a decrease in pHi in spermatogenic cells. This glucose-induced pHi decrease occurred later than the changes in [Ca2+]i, which were also observed when the pHi decrease was inhibited, indicating that the glucose-induced [Ca2+]i increase was not a consequence of pHi changes. Hexose phosphorylation in glycolysis was part of the mechanism by which glucose metabolism induced a [Ca2+]i increase in spermatogenic cells. The sensitivity of [Ca2+]i to carbohydrate metabolism was higher in round spermatids than in pachytene spermatocytes. Thus, differentiation-related changes in carbohydrate metabolism in spermatogenic cells determine a dynamic and differential modulation of their [Ca2+]i by glucose and lactate, two substrates secreted by the Sertoli cells.