<jats:title>ABSTRACT</jats:title> <jats:p> In immunosuppressed hosts, mucormycosis is a life-threatening infection with few treatment options. We studied the activity of colistin (polymyxin E) against <jats:italic>Mucorales</jats:italic> species <jats:italic>in vitro</jats:italic> and in a murine model of pulmonary <jats:italic>Rhizopus oryzae</jats:italic> infection. Colistin exhibited fungicidal activity <jats:italic>in vitro</jats:italic> against <jats:italic>Mucorales</jats:italic> spores and mycelia. At the colistin MIC, initial <jats:italic>R. oryzae</jats:italic> hyphal damage was followed by rapid regrowth; however, regrowth was prevented by combining colistin with a subinhibitory concentration of amphotericin B. Using electron microscopy and FM4-64 staining, we demonstrated that colistin disrupts <jats:italic>R. oryzae</jats:italic> cytoplasmic and vacuolar membranes, resulting in the leakage of intracellular contents. The prophylactic intranasal treatment of immunosuppressed mice with colistimethate significantly reduced the mortality rate and pulmonary fungal burden resulting from inhalational challenge with <jats:italic>R. oryzae</jats:italic> spores, whereas intraperitoneal colistimethate treatment had no effect. We conclude that colistin has modest <jats:italic>in vitro</jats:italic> and <jats:italic>in vivo</jats:italic> fungicidal activity against <jats:italic>Mucorales</jats:italic> spp. Further studies are warranted to assess the use of this drug in the prevention and treatment of mucormycosis.