Bcl-x exists in two isoforms, the anti-apoptotic form Bcl-xL and the proapoptotic form Bcl-xS. The critical balance between the two forms appears to be important for cell survival; however, it is still not clear exactly how the vital balance is maintained. Using an in vitro spermatogenic cell apoptosis model, this study provides a new insight into the possible role of Ca2+ in regulating the Bcl-xS and Bcl-xL expression. 2,5-Hexanedione, a metabolite of the common industrial solvent n-hexane, caused a significant increase in reactive oxygen species followed by an enhancement of intracellular Ca2+ through the T-type Ca2+ channels. Consequent to the above changes, expression of Bcl-xS increased with a concomitant drop in Bcl-xL expression, thus altering the ratio of the two proteins. Impediment of Ca2+ influx by using a T-type Ca2+ channel blocker pimozide resulted in a decrease in Bcl-xS and an increase in Bcl-xL expression. This caused prevention of mitochondrial potential loss, reduction of caspase-3 activity, inhibition of DNA fragmentation, and increase in cell survival. Alternatively, Ca2+ ionophores caused an increase of Bcl-xS encoding isoform over the Bcl-xL-encoding isoform. Therefore, this study proposes a role for Ca2+ in regulation of Bcl-xS and Bcl-xL expression and ultimately cell fate.