Membrane morphology is an important structural determinant as it reflects cellular functions. The pentaspan membrane protein Prominin-1 (Prom1/CD133) is known to be localised to protrusions and plays a pivotal role in migration and the determination of cellular morphology; however, the underlying mechanism of its action have been elusive. Here, we performed molecular characterisation of Prom1, focussing primarily on its effects on cell morphology. Overexpression of Prom1 in RPE-1 cells triggers multiple, long, cholesterol-enriched fibres, independently of actin and microtubule polymerisation. A five amino acid stretch located at the carboxyl cytosolic region is essential for fibre formation. The small GTPase Rho and its downstream Rho-associated coiled-coil-containing protein kinase (ROCK) are also essential for this process, and active Rho colocalises with Prom1 at the site of initialisation of fibre formation. In mouse embryonic fibroblast (MEF) cells we show that Prom1 is required for chloride ion efflux induced by calcium ion uptake, and demonstrate that fibre formation is closely associated with chloride efflux activity. Collectively, these findings suggest that Prom1 affects cell morphology and contributes to chloride conductance.