The rapeseed crop (Brassica napus) is valued mainly for animal feed, food oil and biofuel production. This plant is particularly susceptible to many pathogens like parasitic plants, fungi or animals attacking aerial or root parts. Conventional plant protection products, used intensively in agriculture, have a negative impact on the environment (air and water quality, biodiversity, etc.) as well as on human health. There is therefore a growing demand for the development of new, more planet-friendly alternative protection methods such as biocontrol compounds. Natural rhamnolipids (RLs) can be used as elicitors of plant defense mechanisms. Indeed, these glycolipids, from bacteria secretome, are biodegradable, non-toxic and they are known for their stimulating and protective effects, in particular on rapeseed against filamentous fungi. Characterizing the organ responsiveness to defense stimulating compounds like RLs is missing. This analysis is crucial in the frame of optimizing the effectiveness of RLs against various diseases. A TMT labeling of the proteins extracted from the shoots and roots of B. napus has been performed and showed a differential pattern of protein abundance between them. These results have allowed identifying several protein families possibly involved in the differential responses observed in shoots and roots of rapeseed upon RLs treatment. In particular, quantitative proteomic analysis highlighted differential accumulation of parietal and cytoplasmic defense or stress proteins in response to treatments with RLs with a clear effect of the type of application (foliar spraying or root absorption). These results must be considered for further use of RLs to fight specific rapeseed pathogens.