In this study, we used two-dimensional gel electrophoresis and MALDI-Q-TOF-MS/MS analysis to examine the global protein expression of a pair of colorectal carcinoma cell lines, SW620 and irinotecan-resistant SW620. Of the 30 spots identified as differentially expressed proteins (±over twofold, P<0.05) between the two cell lines, 26 spots (corresponding to 26 unique proteins) were positively identified by MALDI-Q-TOF-MS/MS analysis. These proteins could be grouped into main classes including metabolism (15.38%), cell SSproliferation/differentiation (11.53%), molecular chaperone (11.53%), mRNA splicing (11.53%), and so on. The proteins, which might be involved in the development of tumor drug resistance, such as α-enolase, cofilin, and thioredoxin-dependent peroxide 1, have been validated by western blot analysis and have been discussed. The proteins identified in this study may be useful in showing the mechanisms underlying irinotecan resistance.