The herbs with sulfur-fumigation may induce chemical transformation thus causing harmful effects on patients. In the current study, the difference of physicochemical property from sulfur-fumigated Smilax glabra Roxb. polysaccharides (SSGRP) and non-fumigated Smilax glabra Roxb. polysaccharides (NSGRP) were characterized and compared, such as external appearance, dissolvability, extraction yield, glucose content, inorganic elements analysis, UV and IR scanning spectrum. Additionally, the immunotoxicity and mechanisms of SSGRP and NSGRP on immune response of murine abdominal RAW264.7 macrophage cells were evaluated by cell viability, flow cytometry, quantitative real-time PCR and western blotting analyses. The results demonstrated that NSGRP could not affect the proliferation of RAW264.7 cells but SSGRP could effectively inhibit the cells viability by inducing apoptosis. SSGRP could also up-regulated the mRNA expression of apoptosis factors including Box and caspase-8. Further investigation elucidated that NSGRP exhibited excellent immunomodulatory activity of RAW264.7 cells, however, SSGRP might inhibit the activity through down-regulating the tumor necrosis factor-a (TNF-alpha) and interleukin-6 (IL-6) mRNA expression as well as blocking the phosphorylation of phosphorylated extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK). In conclusion, our study suggested that sulfur-fumigation displayed significant immune toxicity on immune response of murine abdominal RAW264.7 macrophages, and the study provided new insights in controlling the sulfur-fumigation processing and storage method in Chinese herbal medicines.