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We report a proteomic analysis of microdissected material from formalin‐fixed and paraffin‐embedded colorectal cancer, quantifying >7500 proteins between patient matched normal mucosa, primary carcinoma, and nodal metastases. Expression levels of 1808 proteins changed significantly between normal and cancer tissues, a much larger fraction than that reported in transcript‐based studies. Tumor cells exhibit extensive alterations in the cell‐surface and nuclear proteomes. Functionally similar changes in the proteome were observed comparing rapidly growing and differentiated CaCo‐2 cells. In contrast, there was minimal proteomic remodeling between primary cancer and metastases, suggesting that no drastic proteome changes are necessary for the tumor to propagate in a different tissue context. Additionally, we introduce a new way to determine protein copy numbers per cell without protein standards …