Harpins are heat-stable, glycine-rich proteins secreted by Gram-negative bacteria, which induce hypersensitive response (HR) in non-host plants. In this study, the thermal unfolding and aggregation of harpin Pss from Pseudomonas syringae pv. syringae have been investigated by biophysical approaches. Differential scanning calorimetric studies indicate that thermal unfolding of harpin Pss is a complex process involving three distinct transitions. CD spectroscopy revealed that the secondary structure of the protein, which is predominantly α-helical, remains unchanged until the onset of transition 2, above which the α-helical content decreases while the β-sheet content increases. Dynamic light scattering measurements yielded the hydrodynamic radius (R h ) of harpin Pss as room temperature as 20.54 ± 6.19 nm, which decreases to 9.35 nm at 61 °C. These results could be explained in terms of the following thermal unfolding pathway for harpin Pss : oligomer → dimer → partially unfolded dimer → unfolded monomer. Sequence analysis indicated the presence of at least two leucine-zipper-like motifs in harpin Pss and several other harpins, whereas computational modelling studies suggest that most of them are located on helices present on protein surfaces, suggesting that they can take part in the formation of oligomeric aggregates, which may be responsible for HR elicitation by harpins and their high thermal stability.