Paramphistomosis, caused by the rumen fluke, Calicophoron daubneyi, is a parasitic infection of ruminant livestock which has seen a rapid rise in prevalence throughout Western Europe in recent years. Following ingestion of metacercariae (parasite cysts) by the mammalian host, newly-excysted juveniles (NEJs) emerge and invade the duodenal submucosa which causes significant pathology in heavy infections. The immature larvae then migrate upwards, along the gastrointestinal tract, and enter the rumen where they mature and begin to produce eggs. Despite their emergence, and sporadic outbreaks of acute disease, we know little about the molecular mechanisms used by C. daubneyi to establish infection, acquire nutrients and to avoid the host immune response. Here, transcriptome analysis of four intra-mammalian life-cycle stages, integrated with secretome analysis of the NEJ and adult parasites (responsible for acute and chronic disease respectively), revealed how the expression and secretion of selected families of virulence factors and immunomodulators are regulated in accordance with fluke development and migration. Our data show that whilst a family of cathepsins B with varying S2 sub-site residues (indicating distinct substrate specificities) are differentially secreted by NEJs and adult flukes, cathepsins L and F are secreted in low abundance by NEJs only. We found that C. daubneyi has an expanded family of aspartic peptidases, which is up-regulated in adult worms, although they are underrepresented in the secretome. The most abundant proteins in adult fluke secretions were helminth defence molecules (HDMs) that likely establish an immune environment permissive to fluke survival and/or neutralise pathogen-associated molecular patterns (PAMPs) such as bacterial lipopolysaccharide in the microbiome-rich rumen. The distinct collection of molecules secreted by C. daubneyi allowed the development of the first coproantigen-based ELISA for paramphistomosis which, importantly, did not recognise antigens from other helminths commonly found as co-infections with rumen fluke.