Nonylphenol (NP) is a representative endocrine disruptor that has an adverse effect on male reproductionand posses direct hazard to Sertoli cells, but the mechanism remains incompletely elucidated. Inthe present study, based on the structural comparability and high affinity between NP and membranephospholipid molecules, we tested the hypothesis that entrance of NP into Sertoli cells would alter membranebiophysical characteristics and biochemical functions. First, we used gas chromatography-massspectrometry (GC-MS) to investigate the distribution and pharmacokinetics of NP in Sertoli cells withthe result revealing that NP could penetrate plasma membrane of Sertoli cells. Meanwhile, Sertoli cellstreated with NP exhibited abnormal membrane potential; that is an early depolarization following shorttreatment and hyperpolarization after longer treatment with the highest concentration of NP. Studieson the membrane dynamics indicated that the NP exposure rendered increased membrane fluidity anddecreased microviscosity and molecular order. The result of lactate dehydrogenase (LDH) leakage assaydemonstrated that NP increased membrane permeability in time–dose-dependent manners. Atomic forcemicroscopic (AFM) imaging was applied to examine the membrane topography, and the images showedthat NP treatment caused disturbance of membrane topography. The activities of plasma membrane Ca2+-ATPase, Ca2+-Mg2+-ATPase and Na+-K+-ATPase were also changed following NP exposure. However, FSHreceptor as an important membrane protein was not significantly altered. All the above changes led tothe disturbed intracellular Ca2+ homeostasis which was an important signal triggering apoptosis. Hence,cellular membranes represented a plausible target for NP-induced cytotoxicity.