Nonylphenol (NP), an endocrine disrupting chemical, is an environmental contaminant and many notorious effects on male fertility have been reported in animal models and wild type species. Here, we evaluated the effects of NP in follicle-stimulating hormone (FSH) signal transduction pathways and lipid metabolism using an in vitro model of rat Sertoli cells (SC) primary culture. Results show that an acute (1 h) SC-exposure to NP (10 µM) increased the intra- and extracellular concentrations of free fatty acids (FFA), mainly arachidonic acid (20:4n-6). Phosphatidylinositol seemed to be the major phospholipid source of this 20:4n-6 release by activation of the protein kinase A (PKA)/cytoplasmic phospholipase A2 (cPLA2) pathway. NP also increased diacylglycerols (DAG) levels and the expression (mRNA) of cyclooxygenase 2 (Cox2) and prostaglandin E2 (PGE2) levels. Noteworthy, accumulation of lipid droplets took place after 24 h NP-exposition, which was prevented by both, a PKA and a PLA2 inhibitors. Like FSH, NP triggers the release of 20:4n-6 that is substrate for PGE2 synthesis via PKA/PLA2 activation. In addition, NP induces the formation of DAG that could be required as a cofactor of the PKC-mediated activation of the Cox2 inflammatory pathway. Our findings suggest that NP alters lipid homeostasis in SC by inducing the activation of pro-inflammatory pathways that may trigger adverse effects in testis physiology over time. Concomitantly, the SC enhances the acylation of surplus free fatty acids (including 20:4n-6) in neutral lipids as a protective mechanism to shield itself from lipotoxicity and pro-inflammatory signals.