The migration of the trematode parasite Fasciola hepatica within its vertebrate host following infection by ingestion of the metacercariae represents a critical event in its establishment and survival. The early stages of infection, during which F. hepatica crosses the intestinal barrier and advances to the liver through the peritoneum, initiate changes in the parasite that drive its development from a free-living state on pasture to an obligate blood-feeding parasite. Using an in vivo mouse model, this study explores the proteomic changes in the parasite as it crosses the intestinal barrier and migrates to the peritoneal cavity (24 h post infection (p.i.)) and liver parenchyma (8 days p.i.). This model was coupled with SWATH-MS, enabling a comparative evaluation of parasite protein abundance during the early stages of infection. We identified a total of 1180 F. hepatica proteins from three developmental time points: newly excysted juveniles (FhNEJ) at 3 h post excystment in vitro, and parasites collected in vivo at 24 h and 8 days p.i., separated into two different parasite compartments (somatic and tegumental). These extracts exhibited differentially expressed proteins (DEPs) across the analyzed time points, with 274 and 463 differentially expressed proteins identified in parasites obtained at 24 h and 8 days p.i., respectively. Our findings further highlight the adaptations F. hepatica undergoes within the first week of infection, including a shift toward anaerobic metabolic pathways, induction of signal transduction pathways involved in growth, and enrichment of crucial cysteine peptidases associated with feeding and immunomodulation. This study represents the first in-depth proteome analysis of parasites recovered 8 days into infection, adding to the wealth of molecular data available for Fasciola spp. to enhance our understanding of early host-parasite interactions. These data are crucial for the development of future in vitro models of fasciolosis and for identifying vaccine candidates targeting the early parasite stages.