Objective To explore the anti-tumor immune function of the Tibetan medicine Rhodiola rosea L. (RRL). Methods Lewis lung cancer-bearing mice were randomly divided into normal saline group, 500 mg/kg RRL ethanol extract treatment group, and 10 mg/kg cyclophosphamide (CTX) treatment group. All the groups underwent the treatment for 10 days. The mouse survival rate and tumor inhibitory rate were calculated. Additionally, the numbers of CD4+T and CD8+ T cells of tumor infiltrating lymphocytes as well as the proportion of FOXP3+ regulatory T cells (Tregs) in the CD4+CD25+Tregs were detected by flow cytometry. Besides, the serum levels of interleukin 2 (IL-2) and γ-interferon (IFN-γ) in the tumor-bearing mice were examined through ELISA, and the spleen cytotoxic T lymphocytes (CTL) activity was detected by the lactic dehydrogenase (LDH) release assay. Results Lewis tumor-bearing mice treated with the ethanol extract of RRL showed remarkably enhanced survival rate and inhibited tumor growth. Furthermore, the number of tumor infiltrating CD4+T and CD8+ T cells increased, while the proportion of FOXP3+ Tregs in the CD4+CD25+Tregs showed a declined tendency. Meanwhile, the serum IFN-γ and IL-2 levels in Lewis tumor-bearing mice increased, and the killing capacity of spleen CTL was enhanced. Conclusion The ethanol extract of RRL has a positive role in enhancing the anti-tumor immunity by regulating the number and function of immunocytes.