Exosomes are secreted nanovesicles consisting of biochemical molecules, including proteins, RNAs, lipids, and metabolites that play a prominent role in tumor progression. In this study, we performed a label-free proteomic analysis of exosomes from a pair of homologous human colorectal cancer cell line with different metastatic abilities. A total of 115 exoDEPs were identified, with 31 proteins upregulated and 84 proteins downregulated in SW620 exosome. We also detected 30 proteins expressed only in SW620 exosomes and 60 proteins expressed only in SW480 exosomes. Bioinformatics analysis enriched the components and pathways associated with the extracellular matrix, cytoskeleton-related pathways, and immune system changes of colorectal cancer (CRC). Cellular function experiments confirmed the role of SW620 exosomes in promoting the proliferation, migration, and invasion of SW480 cells. Further verifications were performed on six upregulated exoDEPs (FGFBP1, SIPA1, THBS1, TGFBI, COL6A1, and RPL10), three downregulated exoDEPs (SLC2A3, MYO1D, and RBP1), and three exoDEPs (SMOC2, GLG1, and CEMIP) expressed only in SW620 by WB and IHC. This study provides a complete and novel basis for exploring new drug targets to inhibit the invasion and metastasis of CRC.