Display omitted •Catozza rapeseeds are a matrix with a nutraceutical value.•The most extensive GSLs profile of Catozza rapeseeds has been provided.•The best myrosinase inactivation is provided at 80 °C for 30 min in rapeseeds.•Catozza rapeseed extracts are exogenous sources of H2S. Glucosinolates (GSLs), anionic sulfur-containing secondary metabolites of Brassicales order, have shown beneficial effects for the prevention of cardiovascular diseases. This study has focused on HPLC-HESI-MS/MS analysis of intact GSLs in a poorly studied food matrix, Catozza rapeseeds (Brassica rapa L. var. rapa DC.), providing the first qualitative profile of this matrix by the identification of fifteen GSLs and the quantification of the nine most abundant compounds. After the evaluation of different powder granulometries, the best myrosinase inactivation treatment condition was achieved with a 30-minute heating treatment at 80 °C, resulting in the highest GSLs content. Two extraction methods were performed on the rapeseed meal to obtain hydroalcoholic Catozza rapeseed extracts (HCRE) and aqueous Catozza rapeseed extracts (ACRE) showing a concentration-dependent and endothelium-independent relaxing effect in mice aorta by releasing hydrogen sulfide (H2S). This study represents the starting point for further investigation of Catozza rapeseed use for GSLs-based formulations for nutraceuticals purpose.