Gelsolin is a calcium-regulated actin binding protein that severs and caps actin filaments. Gelsolin's severing activity is important for regulating actin filament assembly dynamics that are required for cell motility as well as survival. The majority of in vitro studies of gelsolin have been performed in dilute buffer conditions which do not simulate the molecular interactions occurring in the crowded intracellular environment. We hypothesize that crowding results in greater gelsolin severing activity due to induced conformational changes in actin filaments and/or gelsolin. In this study, we evaluated the effects of crowding on gelsolin-mediated actin filament severing and gelsolin binding to actin filaments in crowded solutions, utilizing total internal reflection fluorescence (TIRF) microscopy and co-sedimentation assays. Our data indicates that the presence of crowders causes a decrease in the rate of gelsolin severing as well as a decrease in the amount of gelsolin bound to actin filaments, with greater effects caused by the polymeric crowder. Despite the severing rate decrease, gelsolin-mediated filament severing is increased in the presence of crowders. Understanding the crowding effect on gelsolin-mediated actin filament severing offers insight into the interactions between gelsolin and actin that occur inside the crowded cytoplasm. (C) 2020 The Authors. Published by Elsevier Inc.