In this research, pullulan was incorporated in protein-based encapsulation matrix in order to assess its cryoprotective effect on the viability of freeze-dried (FD) probiotic Lactobacillus acidophilus NRRL-B 4495. This study demonstrated that pullulan in encapsulation matrix resulted in a 90.4% survival rate as compared to 88.1% for whey protein (WPI) encapsulated cells. The protective effects of pullulan on the survival of FD-encapsulated cells in gastrointestinal conditions were compared. FD WPI-pullulan capsules retained higher survived cell numbers (7.10 log CFU/g) than those of FD WPI capsules (6.03 log CFU/g) after simulated gastric juice exposure. Additionally, use of pullulan resulted in an increased viability after bile exposure. FD-free bacteria exhibited 2.18 log CFU/g reduction, while FD WPI and FD WPI-pullulan encapsulated bacteria showed 0.95 and 0.49 log CFU/g reduction after 24 h exposure to bile solution, respectively. Morphology of the FD microcapsules was visualized by scanning electron microscopy.