Intramuscular fat (IMF) is an important trait influencing meat quality, and preadipocyte differentiation is a key factor affecting IMF deposition. Here we compared the transcriptome profiles of porcine intramuscular and subcutaneous preadipocytes during differentiation to gain insight into specific molecular and cellular events associated with intramuscular stromal vascular cell (MSVC) differentiation. RNA-Seq was used to screen for differentially expressed genes (DEGs) during the in vitro differentiation of MSVC and subcutaneous stromal vascular cell (ASVC) on days 0, 2 and 4. A total of 985 DEGs were identified during ASVC differentiation and 1469 DEGs during MSVC differentiation. Among these DEGs, 409 genes were specifically expressed during ASVC differentiation, 893 genes were specifically expressed during MSVC differentiation, and 576 DEGs were co-expressed during ASVC and MSVC differentiation. The expression profiles of DEGs during ASVC or MSVC differentiation were determined by cluster analysis based on Short Time-series Expression Miner (STEM). Four significant STEM profiles (profiles 1, 4, 5, and 14) were determined during ASVC differentiation, and four significant STEM profiles (profiles 1, 4, 11, and 14) were determined during MSVC differentiation. Gene ontology (GO) analysis indicated that DEGs related to adipocyte differentiation were identified to be significantly enriched in both adipose and muscle profile 14. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEGs in adipose profile 14 and muscle profiles 11 and 14 (STEM clustered them into one cluster) showed that the PPAR signaling pathway was significantly enriched in these profiles and four signaling pathways were specifically enriched in muscle profiles 11 and 14. Furthermore, analysis of transcription factor binding sites (TFBS) in the gene set revealed two over-represented transcription factors (NR3C4 and NR3C1), which were specifically significantly enriched in the promoter regions of genes within muscle gene expression profiles 11 and 14.