Low level of drip loss is an important quality characteristic of meat with high economic value. However, the key genes and regulatory networks contributing to drip loss in pork remain largely unknown. To accurately identify the key genes affecting drip loss in muscles postmortem, 12 Duroc × (Landrace × Yorkshire) pigs with extremely high (n = 6, H group) and low (n = 6, L group) drip loss at both 24h and 48h postmortem were selected for transcriptome sequencing. The analysis of differentially expressed genes (DEGs) and weighted gene co-expression network analysis (WGCNA) were performed to find the overlapping genes using the transcriptome data, and functional enrichment and protein-protein interaction (PPI) network analysis were conducted using the overlapping genes. Moreover, we used machine learning to identify the key genes and regulatory networks related to drip loss based on the interactive genes of the PPI network. Finally, nine potential key genes (IRS1, ESR1, HSPA6, INSR, SPOP, MSTN, LGALS4, MYLK2, and FRMD4B) mainly associated with the MAPK signaling pathway, the insulin signaling pathway, and the calcium signaling pathway were identified, and a single-gene set enrichment analysis (GSEA) was performed to further annotate the functions of these potential key genes. The GSEA results showed that these genes are mainly related to ubiquitin mediated proteolysis and oxidative reactions. Taken together, our results indicate that the potential key genes influencing drip loss are mainly related to insulin signaling mediated differences in glycolysis and ubiquitin mediated changes in muscle structure and improves the understanding of gene expression and regulation related to drip loss and contributes to future molecular breeding for improving pork quality.