This paper evaluated the benefit of the polysaccharides from Pholiota Nameko (PNPS-1) as a cryoprotectant for maintaining the viability of Lactobacillus casei ATCC 334 during freeze-drying and subsequent storage. The data showed that all the freeze-dried L. casei ATCC 334 samples added with PNPS-1 conformed to the three evaluation indicators of residual viability (>107 CFU/g), moisture content (<7% wet basis) and water activity (<0.6), which were usually used as the quality standards of the freeze-dried powdered starter products. At the same adding concentration of 0.04 mg/ml, PNPS-1 showed only a little weaker than skim milk in the protection for L. casei ATCC 334. However, the combined cryoprotectants contained 0.04 or 0.02 mg/ml PNPS-1 were significantly higher than 0.04 mg/ml skim milk. PNPS-1 could improve the membrane fluidity and membrane integrity compared with the blank control sample. In addition, acid tolerance, bile tolerance and heat tolerance of the freeze-dried cells were all ameliorated by the PNPS-1 protection. This study revealed the novel application of PNPS-1 as a new cryoprotective agent for the freeze-dried L. casei ATCC 334 preservation.