Probiotic bacterium Escherichia coli Nissle 1917 (EcN) is a Gram-negative probiotic bacterium found in the human intestine. Related researches have shown that EcN has good effects in regulating host immune function and treating intestinal diseases. Compared with EcN probiotics themselves, EcN-derived outer membrane vesicles (OMVs) are more likely to spread, cross the mucus layer, and migrate directly to the intestinal submucosa tissue to interact with cells of the host immune system. EcN-derived OMVs play an important role in intestinal immune regulation. However, there are relatively few studies on the regulation of intestinal immunity by EcN-derived OMVs of different sizes. In this project, we first isolated EcN-derived OMVs and then conducted proteomic analyses on the isolated EcN-derived OMVs. Second, we used the filtration method to obtain EcN-derived OMVs of three specific sizes: < 50 nm, 50–100 nm, and 100–300 nm. We then conducted proteomic analyses on EcN-derived OMVs of three specific sizes.