Updated project metadata. The excretory-secretory (ES) proteins are the molecules that actively or passively released by parasite at the parasite-host interface to maintain a physiological and immunological relationship with the host. In the present study, we characterised the excretory-secretory products from the short-term in vitro culture (i.e. within 12 h) of Haemonchus contortus (as the most important parasitic nematodes of livestock animals worldwide) using a high throughput tandem mass-spectrometry underpinned by the most recent genomic dataset. Totally, 878 unique ES proteins from three key developmental stages were identified and quantified without intracellular protein contamination (e.g. histone) typically occurred in longer term maintenance cultures. Bioinformatic analyses showed noteworthy protein excretion and secretion alterations during the transition from the free-living to the parasitic phase, especially for ES proteins which are likely involved in nutrient digestion, acquisition and parasite-host interactions, such as proteolytic cascade related peptidases, glycoside hydrolases, C-type lectins, transthyretin-like proteins and SCP/TAPS proteins. Our finding provide an avenue to better explore interactive processes between the host and this highly significant parasitic nematode, to underpin the search for novel drug and vaccine targets.