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This study was aimed to determine the effect of CaCl2 on the motility and viability of stallion spermatozoa during different incubation temperatures. Experimental samples were prepared by diluting the ejaculates (n = 10) from three uniformly housed and fed breeding stallions with six different concentrations of CaCl2 (A: 0.1125, B: 0.225, C: 0.45, D: 0.938, E: 1.25, and F: 1.875 mg/mL). The control samples (CON) were prepared by diluting ejaculate only with physiological solution. Samples were divided into two aliquots for analyses at different storage temperatures (5 °C and 37 °C). The motility parameters were analysed by Computer Assisted Semen Analysis system at several time intervals (0, 1, 2 and 3 h) and the viability was assessed using a mitochondrial toxicity test (MTT) realized at the end of incubation at both temperatures. Addition of CaCl2 to stallion semen showed significant effect on motility parameters, especially in the highest concentrations at 5 °C. Significant objectionable effect of CaCl2 on both total and progressive motility was observed at temperature 37 °C compared to control sample. However, results of velocity curved line in samples C, D and F at time 1 h and also at time 2 h in sample F showed significant positive effect of CaCl2. Sperm viability in experimental samples did not show a significant difference compared to the control at either 5 °C or 37 °C. The results of this study did not confirm essential effect of calcium on reproductive parameters of stallion. To conclude, our study demonstrated that the effect of CaCl2 on stallion sperm motility differs in a dose-dependent manner; however, the overall impact on motility parameters does not seem to be beneficial.