Electrospray ionization (ESI) is considered a soft ionization method, and typically no peptide ion fragmentation is observed. Recently, it has been observed that intensive fragmentation of peptide ions can occur during nanoelectrospray (nanoESI) at special conditions, such as solutions containing high concentrations of salt and relatively low voltage for the spray. In this study, peptide fragmentation during nanoESI has been systematically characterized. The fragmentation phenomenon was observed for a variety of peptides with molecular weights lower than 3000 Da and the types of fragments include a, b, and y ions. For phosphorylated peptides, very little loss of the labile phosphate groups was observed. Solution electrical conductivity (K) and flow rate were identified as the key parameters affecting the degree of peptide fragmentation. Mechanistic studies suggested that very fine first generation charged droplets (∼30 nm in diameter) with high surface electric field (∼1 V/nm) could be formed from nanoESI of highly conductive solutions (K = 0.4 S/m) and at a low flow rate (2 nL/min). It is proposed that solvated peptide ions are ejected with high kinetic energies from the early generations of charged droplets, and the subsequent collisional activation in air induces the peptide fragmentation. The relatively high degree of solvation around the phosphate groups may contribute to the preservation of the phosphorylation during the activation process.