Patients with metastatic colorectal cancer (mCRC) carrying BRAFV600E mutation have worse response to chemotherapy and poor prognosis. The BRAFV600E inhibitor vemurafenib has shown modest efficacy as monotherapy in BRAF-mutated mCRC due to the development of resistance. The aim of this study was to conduct a comparative proteomics profiling of the secretome from vemurafenib-sensitive vs. -resistant colon cancer cells harboring BRAFV600E mutation in order to identify specific secretory features potentially associated with changes in the resistant cells’ phenotype. Towards this aim, we employed two complementary proteomics approaches including two-dimensional gel electrophoresis coupled with MALDI-TOF/TOF mass spectrometry and label-free quantitative LC-MS/MS analysis. Obtained results pointed to aberrant regulation of DNA replication and endoplasmic reticulum stress as the major secretome features associated with chemoresistant phenotype. Accordingly, two proteins implicated in these processes including RPA1 and HSPA5/GRP78 were discussed in more details in the context of biological networks and their importance as potential secretome targets for further functional and clinical evaluation. Expression patterns of RPA1 and HSPA5/GRP78 in tumor tissues from colon cancer patients were also found in additional in silico analyses to be associated with BRAFV600E mutation status, which opens the possibility to extrapolate our findings and their clinical implication to other solid tumors harboring BRAFV600E mutation, such as melanoma.