Purpose <p>The purpose of this study was a proteomic investigation of test enamel of HSPM vs the control enamel of Second Primary Molars, to gain an insight into the possible mechanism of pathogenesis of hypomineralisation.</p> Methods <p>Enamel sample blocks were sectioned and processed for label-free quantitation of proteins by LC–MS/MS, followed by bioinformatics analyses of differentially expressed proteins (DEP); two DEPs were validated by western/dot blot.</p> Results <p>There was a significant elevation of total number of proteins in the HSPM samples as compared to the control enamel. A significant elevation of proteins such as protein LEG1 homolog, Odontogenesis-associated phosphoprotein, Leucocyte elastase inhibitor, Olfactomedin-like protein 3 and albumin were observed in HSPM-affected enamel, along with upregulation of calcium-binding proteins and immunoglobulins. Evaluation of signalling pathways contributed by the upregulated proteins in HSPM enamel samples was suggestive of increased levels of different components of the immune (defence) system such as raised antibodies (immunoglobulins level), complement coagulation cascade activation, neutrophil and platelet degranulation as well as defence response to bacteria/ fungus.</p> Conclusions <p>The study results suggest the possibility of an underlying infectious/inflammatory aetiology responsible for hypomineralisation of primary enamel in these patients. Such a proteomics approach is useful to guide preventive interventions for HSPM.</p>