Molting is a key step for body-size expansion and environmental adaptation of parasitic nematodes, and it is extremely important for Trichinella spiralis growth and development, but the molting mechanism is not fully understood. In this work, label-free LC-MS/MS was used to determine the proteome differences between T. spiralis muscle larvae (ML) at the encapsulated stage and intestinal infective larvae (IIL) at the molting stage. The results showed that a total of 2885 T. spiralis proteins were identified, 323 of which were differentially expressed. These proteins were involved in cuticle structural elements, regulation of cuticle synthesis, remodeling and degradation, and hormonal regulation of molting. These differential proteins were also involved in diverse intracellular pathways, such as fatty acid biosynthesis, arachidonic acid metabolism, and mucin type O-glycan biosynthesis. qPCR results showed that five T. spiralis genes (cuticle collagen 14, putative DOMON domain-containing protein, glutamine synthetase, cathepsin F and NADP-dependent isocitrate dehydrogenase) had significantly higher transcriptional levels in 10 h IIL than ML (P < 0.05), which were similar to their protein expression levels, suggesting that they might be T. spiralis molting-related genes. Identification and characterization of T. spiralis molting-related proteins will be helpful for developing vaccines and new drugs against the early enteral stage of T. spiralis.