The aim of this study is to explore the mechanism by which acrolein (ACR), a metabolite of cyclophosphamide (CP), induces immature Sertoli cell cytoskeletal changes. Sertoli cells obtained from rats were cultivated and treated with 50 and 100 μM ACR. XTT assays were performed to detect cell viability. Activities of superoxide dismutase (SOD), glutathione peroxidases (GSH-Px), and catalase (CAT), as well as total anti-oxidation competence (T-AOC) were examined. Superoxide anion levels were detected by a fluorescent probe. Cell ultrastructure changes were observed by transmission fluorescent microscope. Actin filament (F-actin) distribution was detected by immunofluorescence, and ERK and p38MAPK expression were detected by western blot analysis. ACR significantly decreased the viability of Sertoli cells in a dose- and time-dependent manner. T-AOC and the antioxidant activity of SOD, CAT and GSH-Px, were decreased in ACR-treated groups compared with the control group. The levels of reactive oxygen species (ROS) in ACR-treated Sertoli cells were increased. In addition, characteristics of cell apoptosis such as mitochondrial swelling, aggregated chromatin, condensed cytoplasm, nuclei splitting, and nuclei vacuolization were observed in ACR-treated cells. Furthermore, ACR-treatment also induced microfilament aggregation, marginalization and regionalization. The expression levels of ERK and p38MAPK were also increased in ACR-treated cells in a dose- and time-dependent manner. ACR, a major CP metabolite, impairs the cytoskeleton which is likely caused by induction of the oxidative stress response through up-regulation of ERK and p38MAPK expression.