The purpose of this study was to determine the effects of 2,4-D, an herbicide used worldwide also known as endocrine disruptor, in Sertoli cell (SC) metabolism. Immature rat SCs were maintained 50h under basal conditions or exposed to 2,4-D (100nM, 10μM and 1mM). SCs exposed to 10μM and 1mM of 2,4-D presented less intracellular glucose and lactate content. Exposure to 10μM of 2,4-D induced a significant decrease in glucose transporter-3 mRNA levels and phosphofructokinase-1 mRNA levels decreased in cells exposed to 100nM and 10μM of 2,4-D. Exposure to 100nM and 10μM also induced a decrease in lactate dehydrogenase (LDH) mRNA levels while the LDH protein levels were only decreased in cells exposed to 1mM of 2,4-D. Exposure to 2,4-D altered glucose uptake and metabolization in SCs, as well as lactate metabolism and export that may result in impaired spermatogenesis.