Cytokines and growth factors are thought to contribute to skeletal muscle hypertrophy. Leukemia inhibitory factor (LIF), a cytokine, enhances skeletal muscle regeneration; however the role of LIF in skeletal muscle hypertrophy remains uncertain. We examined the hypertrophic ability of the plantaris and soleus muscles in wild-type mice (WT) and LIF knock-out mice [LIF(−/−)] in response to increased mechanical load. Using the functional overload model to induce increases in mechanical load on the plantaris and soleus muscle, WT mice demonstrated increases in plantaris and soleus mass after 7, 21, and 42 days of loading. However, the LIF(−/−) mice had no significant increases in plantaris muscle mass at any time point, while the soleus muscle exhibited a delayed hypertrophic response. Systemic delivery of LIF to the LIF(−/−) mice returned the hypertrophic response to the same levels as the WT mice after 21 days of functional overload. These data demonstrate for the first time that LIF expression in loaded skeletal muscle is critical for the development of skeletal muscle hypertrophy in the functional overload model.