Abstract: Localization of normal microflora in the GIT of ruminants is an important factor, which enhances evolution of their digestive system. Probiotics are one of these options, which contribute in this manner. The present study evaluated single or twice daily milk feeding with and without probiotic on daily calves` performance. Forty Holstein calves weighing 43±5 kg were used in a completely randomized design test to study the effects of probiotic on food intake, blood characteristics and coliform bacteria population of feces. Treatments were as follows: T1) control with feeding four liters of milk twice daily, T2) probiotic with feeding four liters of milk twice daily, T3) control with feeding four liters of milk once daily, T4) probiotic with feeding four liters of milk once daily. From first day to end of the experiment (49 days) T2 and T4 received one-gram probiotic in their milk daily. Water and calf starter were offered free choice. Calves were weighed weekly. Intakes of starter were recorded daily and jugular blood sample were taken every 10 days for recording albumin (A), IgA, IgG and A/G ratio. Feces samples were taken every two weeks. Despite findings in earlier reports the results of this experiment did not confirm the positive effect of probiotic on calf performance. Feed intake mean for calves in treatment 2, was 370.8 g and for treatment 1 was 351.8 g. Body weight gain for group with probiotic (T2, T4) and control (T1, T3) was 441.5 and 422.8 g day-1, respectively. Feed efficiency for probiotic treatments and control groups was 1.3 and 1.4 g g-1, respectively. There was no significant difference (p>0.05) between probiotic and non-probiotic groups for any of the performance parameters recorded. Feed intake, body weight gain and Feed efficiency for calves feeding milk once daily and twice daily was 372.4 and 348.8 g, 381.6 and 480.9 g day-1, 1 and 1.4 g g-1, respectively. Feed intake was not significantly different (p>0.05) between groups with feeding milk once or twice daily, but body weight gain and feed efficiency were significantly (p<0.05) different between the treatments. In conclusion, probiotic used in this study did not affect the performance of dairy calves, but feeding milk once or twice daily influenced their performance.