Leukemia inhibitory factor (LIF) plays important roles in varieties of biological processes. This factor is highly conserved in mammalian animals and only one heterozygous LIF mutation was reported to cause the infertility of women. A LIF mutation was generated and the evidences were provided that the mutation of mature LIF at the 29th amino acid totally abolished its functions, including stimulation of STAT activation assayed by Luciferase reporter gene expression and EMSA experiments. In addition, the mutated LIF failed to inhibit the proliferation of M1 cells. The data indicated that the mutation of LIF did not have a dominant negative effect but lost the biological functions, suggesting that the 29th amino acid is critical for maintaining the activities of LIF.