Extracellular vesicles (EVs) are nano sized membrane-bound messengers released by most cells, serving as pivotal mediators in intercellular communication. There is a substantial body of evidence that imply many cancer cell types release higher amounts of EVs compared to healthy cells. However, molecular machinery in the biogenesis of EVs remains poorly understood. TMEM16F, a calcium activated phospholipid scramblase that has been implicated in the formation of vesicles from the plasma membrane, known as micro vesicles, under specific stimulatory conditions. However, little work has been done under non-stimulatory conditions. Hence, the importance of TMEM16F in EV biogenesis and function at basal levels was studied. In our study, we generated three TMEM16F KO cancer cell lines using CRISPR/Cas9 and used various biochemical approaches to understand the biogenesis and function of these EVs. Our results showed that TMEM16F-deficient cells released small EVs (sEV) of comparable size to those from wildtype (WT) counterparts, but at significantly reduced numbers. Interestingly we showed that, EVs lacking TMEM16F we functionally altered and were not equal in their ability to promote growth and colony formation in a carcinoma cell setting. To further understand the mechanism behind the perturbation, we conducted proteomic and lipidomic analysis, revealing that loss of TMEM16F modified the protein and lipid cargo of EVs. Our findings suggest a novel mechanism for TMEM16F in regulation EV biogenesis and function under basal condition across multiple carcinoma models. These findings provide new insight into how EV dysregulation may influence cancer progression and highlight TMEM16F as a potential therapeutic target.