Extracellular vesicles (EVs) such as exosomes are loaded with specific biomolecules in order to perform cell-to-cell communication. Understanding the mechanism of selective cargo loading is important to better understand the physiological and pathological function of EVs. Here we describe a novel target of the E3 ligase TRIM25 and show that inflammation-mediated EV loading of the RNA binding protein FMR1 and its associated microRNA, miR-155, is promoted by TRIM25-mediated K63-ubiquitination of FMR1. This ubiquitination promotes an interaction between FMR1 and the EV loading machinery via the cleavage of the trafficking adaptor protein RILP. These interactions are lost when TRIM25 is knocked down. Loss of TRIM25 also prevents the loading of both FMR1 and miR-155. These findings suggest that inflammation-mediated loading of FMR1 and its associated microRNAs into the EV are dependent on K63-ubiquitination by TRIM25 and provide novel insights and tools to manipulate EV biogenesis for therapeutic benefit.