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The aim of this study was to investigate the effects of Phyllanthus Emblica polysaccharide (PEP) on immunomodulation and intestinal flora in cyclophosphamide (CTX)-treated female mice. The results showed that PEP could directly modulate the immune activity of cyclophosphamide (CTX) in mice by attenuating its damage to spleen, thymus and intestinal tissues. Meanwhile, our study revealed that PEP could activate TLR4-mediated MAPK and NF-κB pathways and promote the expression of cytokines such as IL-2 and TNF-α, thus modulating the immune function of immunosuppressed mice. In addition, PEP was positively correlated with immune properties, with the strongest correlation with spleen index, and PEP could also regulate the immune function of mice by modulating the intestinal flora, such as increasing the content of Lactobacillus and Bifidobacterium. Therefore, PEP may be a potential novel immunomodulator to ameliorate CTX-induced immunosuppression and intestinal flora dysregulation.