Display omitted •Proteomics was used to investigate proteome changes in germinating M. oleifera seeds.•The DEPs within the molecular weight range of 10–60 kDa were most abundant.•A peptidase and three types of endopeptidases were identified during germination.•M. oleifera germinating seeds contained a mixture of milk-clotting proteases.•The highly expressed proteases have potential application in the food industry. This study is the first to apply label-free based proteomics to investigate the proteome changes during Moringa oleifera seed germination. In total, 1267 proteins were identified, with proteins within the molecular weight range of 10–60 kDa being most abundant. Among the 174 differentially expressed proteins (DEPs), 42 were upregulated and 26 downregulated, whereas 45 and 61 were uniquely expressed in the matured dry and germinating seeds, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the main DEPs had significant molecular functions related to catalytic activity and were involved in metabolism, degradation and biosynthesis, with 49 enzymes highly expressed in germination, including 9 proteases with hydrolytic activity, of which a peptidase and three types of endopeptidases were associated with milk-clotting activity. We further demonstrated that proteolytic activity (PA) and milk-clotting specific activity (MCSA) of protease extracts from M. oleifera seeds were increased during germination, particularly for samples from the ammonium sulfate (AS) fractionation that were statistically significance (p value < 0.05). Overall, proteases derived from germinating M. oleifera seeds can be used in the food industry, especially for potential application in the production of bioactive peptides and cheese processing.