Myostatin (MSTN) resulted in reduced backfat thickness in MSTN-knockout (MSTN-KO) pigs, whereas the underlying mechanism remains elusive. In this study, RNA sequencing (RNA-seq) was used to screen differentially expressed genes (DEGs) in porcine fat tissues. We identified 285 DEGs, including 4 adipocyte differentiation-related genes (ADRGs). Matrix Metalloproteinase-2/7 (MMP-2/7), fibronectin (FN), and laminin (LN) were differentially expressed in MSTN-KO pigs compared with wild-type (WT) pigs. To investigate the molecular mechanism, we treated the preadipocytes with siRNA and recombinant MSTN protein. The results indicated that MSTN increased the expression of MMP-2/7/9 and promoted the preadipocyte differentiation. To further validate the effect of MSTN on MMP-2/7/9 expression, we treated MSTN-KO PK15 cells with recombinant MSTN protein and detected the expression of MMP-2/7/9. The data showed that MSTN increases the expression of MMP-2/7/9 in PK15. This study revealed that MSTN promoted preadipocyte differentiation and provided the basis for the mechanism of fatty deposition in pigs.