Secreted proteins constitute a major part of virulence factors that are responsible for pathogenesis caused by gram negative bacteria. Enterohemorrhagic Escherichia coli (E. coli), EHEC O157:H7 is the major pathogen often causing outbreaks. There is growing evidence that non-O157:H7 E. coli strains may also be involved in the recent outbreaks. However, there is no systematic study describing differential secreted proteins from non-O157:H7 E. coli strains. Here, we have applied isobaric tag-based TMT labeling combined with high-resolution Fourier transform mass spectrometry to study the differential secretome analysis of major non-O157:H7 E. coli strains, O103, O111, O121, O145, O26 and O45, which is known as diarrhea inducing non-O175:H7 ATCC “big six” serogroup E. coli strains. We identified 1,240 proteins quantitatively identified, 565 proteins were found to be secreted as predicted by PSORTb and SecretomeP. We identified 310 proteins containing signal peptide and 255 proteins as secreted. We identified 20 strain specific proteins with in big-six group and was confirmed by proteogenomics approach. Further we enriched and have shown relative expression of type III secretion system. To our knowledge, this study is the first comparative proteomic study on secretome of E. coli big six serogroup and the several of these strain specific secreted proteins can be further studied to develop potential markers for identification and strain level differentiation. Moreover, the results of this study can be utilized in several applications, including food safety, diagnostics of E. coli outbreaks, and biodefense.