The major allergen Der p 1 of the house dust mite Dermatophagoides pteronyssinus is a papain-like cysteine protease (CA1) associated to the development of allergic diseases such as asthma, rhinitis or atopic dermatitis. This allergen is expressed as an inactive precursor, called proDer p 1, formed by a 25 kDa catalytic domain downstream to an 10 kDa N-terminal propeptide, which blocks the active site cleft. The propeptide of Der p 1 exhibits a specific fold, which makes it unique in the CA1 propeptide family as it is characterised by the presence of four alpha helices and the absence of ERFNIN motif. In this study, we investigated the activation steps involved in the maturation of recombinant proDer p 1 expressed in Pichia pastoris under acidic conditions and we studied the influence of acidic pH on the structure of both propeptide and catalytic domain. Therefore, we characterized the interaction between the propeptide and mature Der p 1 at different pH values in terms of activity inhibition, structural stability and proteolytic susceptibility. According to our results, the auto-activation of proDer p 1 is a multistep mechanism, characterized by at least two intermediates (ATFE- and SNGG-) corresponding to the loss of the first and second propeptide alpha helices, respectively. The propeptide strongly inhibits unglycosylated and glycosylated recombinant Der p 1 (KD= 7 nM) at neutral pH. This inhibition is pH dependent, decreasing from pH 7 to pH 4 and can be related to structural changes of the propeptide initiated by the protonation of the aspartate residue of Lys17-Asp51-Tyr19 structural triad presents within the propeptide N-terminal domain. This protonation triggers conformational changes of the first propeptide alpha helix leading to an increase of the propeptide flexibility, an increase of its proteolytic sensitivity and the formation of a molten globule state. In addition, we compare mature protease, zymogen and propeptide pH unfolding and stability and highlights that the presence of the propeptide does not influence the catalytic domain pH unfolding and stability as the propeptide displays a weaker pH stability than the protease domain. These results confirmed that the propeptide unfolding is the key event of the activation process. Finally, we unravel the intermolecular contribution of mature Der p 1 in the activation process and highlights that activation of the precursor can be achieved, under acidic conditions, by intermolecular process but initial auto-activation most probably occurs through an intramolecular process or by the proteolysis by the catalytic domain of another zymogen in which the propeptide is unfolded. According to our results, we proposed that activation of the zymogen at pH 4 reflects a compromise between activity preservation and propeptide unfolding and that the location of the activation sites on the propeptide structure is a compromise between sequence recognition specificity and proteolytic susceptibility of the corresponding area.