Interferon (IFN)-lambda, also known as IL-28A, IL-28B, or IL-29, is a new type III IFN, which shares many functional characteristics with type I IFN (alpha/beta). Currently, IFN-alpha is used in the treatment of certain forms of cancer with severe adverse effects. Some researches had stated that IFN-lambda s induced a similar but restricted growth inhibition of tumor cells relative to IFN-alpha; moreover, mutations of IFN-lambda s could strongly impact its biological properties. In this study, three hIL-29 mutants (K33R, R35K, and K33R/R35K) were generated by site-directed mutagenesis and efficiently expressed in Pichia pastoris GS115, which have considerable abilities to inhibit the growth of BEL-7402, HCT-8, and SGC-7901 tumor cells in vitro. The results showed that these mutants (K33R, R35K, and K33R/R35K) exhibited a significantly enhanced anti-proliferation activity against these tumor cells, compared with native hIL-29 in vitro. Further assay in vitro indicated that superior to K33R and R35K, K33R/R35K had a significant increase in anti-tumor activity compared with IFN-alpha 2b, which suggested that the K33R/R35K could make improvement for the effectiveness of native hIL-29 in clinic and could be used as a potentially powerful candidate for cancer immunotherapy.