AbstractMetastatic dissemination of colorectal cancer (CRC), the third most common cancer type, is responsible for CRC deaths. Understanding the transition of lymph node metastasis (LNM) from Stage II to Stage III is beneficial in the prognosis and intervention of CRC. In this study, a quantitative proteomic survey was conducted to investigate the LNM‐associated proteins and evaluate the clinicopathological characteristics of these target proteins in CRC. By using the LC–MS/MS iTRAQ technology, we analysed the proteomic changes between LMN II and LMN III. Fresh tumours from the CRC specimens consisting of 12 node‐negative (Stage II) and 12 node‐positive (Stage III) cases were analysed by LC–MS/MS iTRAQ proteome analysis. Subsequently, tissue microarray with immunohistochemistry staining was conducted to access the clinicopathological characteristics of these proteins in 116 paraffin‐embedded CRC samples, each for non‐LNM and LNM CRC. To study the effects of the differentially expressed proteins on the potential mechanism, Boyden chamber assay, flow cytometry and shRNA‐based assessments were conducted to examine the role of the epithelial–mesenchymal transition (EMT) and the invasiveness of CRC cells and others in vivo xenograft mouse model experiments. Forty‐eight proteins were found differentially expressed between non‐LNM and LNM CRC tissues. Protein abundances of chromogranin‐A (CHGA) and ubiquitin carboxyl‐terminal hydrolase isozyme L1 (UCHL1) were observed in node‐positive CRC (p< 0.05). Knockdown of CHGA and UCHL1 significantly regulate cancer behaviours of HCT‐116, including inhibition of cell migration, invasiveness, cell cycle G1/S arrest and reactive oxygen species (ROS) generation. Mechanistically, the CHGA and UCHL1 inactivation displayed decreased levels of UCH‐L1, chromogranin A, β‐catenin, cyclin E, twist‐1/2, vimentin, MMP‐9, N‐cadherin and PCNA through the activation of the Rho‐GTPase/AKT/NFκB pathways. Histone modification of H3K4 trimethylation of CHGA and UCHL1 promoter were increased to activate their transcription through the signalling transduction such as Rho‐GTPase, AKT and NFκB pathways. Our results indicated that UCHL1 and chromogranin A are novel regulators in CRC lymph node metastasis to potentially provide new insights into the mechanism of CRC progression and serve as biomarkers for CRC diagnosis at the metastatic stage.