Metastasis-associated lung adenocarcinoma transcript 1 (Malat1) is an example of a functional long noncoding RNA involved in many biologic processes. However, the mechanisms for Malat1 in myogenesis are unclear. Serum response factor (SRF) is a pivotal transcription factor for muscle proliferation and differentiation and is reported to be a target gene for muscle-specific microRNA-133 (miR-133). In this study, we initially found that silencing Malat1 in the mouse myoblast C2C12 cell line inhibited myocyte differentiation and decreased Srf at both the RNA and protein levels. Srf silencing decreased Malat1 expression as well. Further study revealed that Malat1 contained an miR-133 functional target site, and the interplay between Malat1 and Srf was miR-133 dependent. We demonstrated that Malat1 modulates Srf through miR-133 as a competing endogenous RNA and established a novel connection among Malat1, miR-133, and Srf in myoblast differentiation.