Colorectal cancer (CRC) invades and metastasizes to local lymph nodes or distal organs in late stages, leading to reduction in survival. Current late stage treatments (chemotherapy and radiotherapy) are often ineffective and/or highly toxic. Targeted and immunotherapies hold promise in the treatment of metastasis. We previously discovered an interaction network in commonly Ras-mutated CRCs, involving urokinase plasminogen activator receptor (uPAR). One particularly novel observation was the direct interaction with the cancer cell-restricted integrin ανβ6 (i.e., uPAR•ανβ6). This interaction has been found to play crucial roles in biologies associated metastasis in a high percentage of patients who develop late stage CRC. Here, we rationally designed interference peptides (iPEPs) modelled on uPAR surfaces involved in uPAR•αvβ6 as potential lead antagonists. CRC SW480β6OE cells expressing uPAR•ανβ6 were treated with biotinylated iPEPs or scrambled peptides, with binding monitored using streptavidin-FITC and cytoskeleton actin changes with phalloidin. This experiment confirmed only iPEP2 and iPEP6 bound and increased actin “spicule” formation. Matrigel invasion and quantitative cellular proteome changes were performed on SW480β6OE (i.e., αvβ6 overexpressing) and naive SW480 cells with/without iPEP treatment. Again, iPEP2 and iPEP6 inhibited growth and invasion, altered morphology, switched key non-canonical MAPK signalling events and significantly changed both the total cell and plasma membrane proteome. A total of 1,358 common proteins were stringently identified across triplicate MS runs between SW480β6OE cells with/without iPEPs, of which 37 were downregulated and 67 significantly upregulated (p<0.05, fold change > 2). Pathway analysis showed 88 cancer-related morphology and signalling-related pathway proteins up/down regulated. uPAR•ανβ6 expression switched signalling to favour MAPK-dominant over SMAD pathways and this could be reversed by iPEPs2/6. The uPAR•ανβ6 interactome drives CRC cells to metastasis and antagonising the interaction disrupts processes associated with proliferation, invasion and growth. iPEPs conceivably represent a therapeutic strategy to curb metastasis.