Infection-inflammation mediated interactions between human ficolin and the pathogen GlcNAc is associated with local acidosis, leading to antimicrobial action. Therefore, revealing the precise molecular conformation induced by pH-shift is crucial in understanding the immune response. Here, we performed constant-pH molecular dynamics simulations on the L-ficolin fibrinogen-like domain over pH 4.5–9. An unusual cis-Asn244-Cys245 peptide bond prevailed over the pH range in the S1 binding site. Analysis of the hydrogen-bond network at S1 suggested Asn244 to be indispensible for maintaining the cis form of Asn244-Cys245, and the absence of the hydroxyl group on Phe262 accounts for the lack of GlcNAc binding.