The fungus Puccinia striiformis f. sp. tritici (Pst), the causal agent of wheat stripe rust, is an obligate biotrophic basidiomycete. Many studies have found that myosins play important roles during fungal growth and propagation. However, there are few reports on the myosins of Pst. In this study, we cloned and obtained the myosin light chain gene PsMLC1 from Pst and characterized its expression. Furthermore, the function of PsMLC1 was identified by mutant complementation. As a result, we found that expression of PsMLC1 in Schizosaccharomyces pombe mostly complemented the defects of the cdc4 mutant, indicating that PsMLC1 belongs to the myosin light chain family member. Expression studies showed that the transcript levels of PsMLC1 little changed before 24 h post inoculation then was suddenly down-regulated during Pst infection of wheat. By using ML-7, we observed that inactivity of PsMLC1 greatly reduced the germination rate of urediniospores. These results suggest that PsMLC1 is essential for the early stages of Pst infection of wheat but unnecessary for the later stages of infection. This work elucidates the function of the myosins in Pst and may provide some theoretical basis for controlling strip rust.