In this article, Ser/Thr ligation(on/off) has been realized to enable N-to-C successive peptide ligations using a salicylaldehyde semicarbazone (SAL(off)) group by in situ activation with pyruvic acid of the peptide SAL(off) ester into the peptide salicylaldehyde (SAL(on)) ester. In addition, a peptide with a C-terminal thioester and N-terminal Ser or Thr as the middle peptide segment can undergo one-pot Ser/Thr ligation and native chemical ligation in the N-to-C direction. The utility of this combined ligation strategy in the N-to-C direction has been showcased through the convergent assembly of a human cytokine protein sequence, GlcNAcylated interleukin-25.