The present study aimed to investigate the proteomic difference between melanosis coli (MC) alone and melanosis coli with colon cancer (MCCC). Protein expression in patients with different diseases was analyzed using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/TOF-MS). A total of 14 protein differences with a confidence level of >95% were found. There were six differences between MC and normal tissues, in which two proteins exhibited upregulated expression levels and four proteins exhibited downregulated expression levels in MC. Furthermore, one protein was expressed only in MC (P<0.05). In addition, there were differences in the expression of eight proteins between MC and MCCC tissues, in which one protein had an upregulated expression in MC tissues and seven proteins had an upregulated expression in MCCC tissues. Furthermore, two proteins were only expressed in MCCC tissues (P<0.05). Eight proteins were identified using mass spectrometry and database search. In conclusion, comparative proteomics accurately displayed the expression differences in eight proteins between MC, MCCC and normal colon tissues.