Linuron is a widely used herbicide; its toxicity on the male reproductive system has been recognized. The current study was designed to explore the molecular mechanism underlying linuron-induced reproductive toxicity. Pregnant rats received daily oral gavage of linuron at the dose of 120mg/kg/d from gestation day (GD)12 to GD17. Tissues from male offspring rats were collected for pathological examination and microarray gene expression profiling. Changes in gene expression were further verified by quantitative real-time RT-PCR. Data showed that linuron-exposed offspring rats had a decreased sperm count (88% of controls) and disrupted acrosome formation. There were evident damages in seminiferous tubules and abnormal morphology in mesenchymal cells in samples from linuron-exposed animals. Microarray analysis indicated that the expressions of testosterone synthesis-associated genes, i.e., Star, P450scc, 3β-Hsd, Abp, Cox7a2, Pcna, p450c17and17β-Hsd were significantly altered by linuron exposure, along with other genes involving in cell proliferation and apoptosis, such as c-myc, S6K, Apaf1, and TSC1. These data indicate that linuron upon entering male offspring body can directly or indirectly interact with the androgen production and function; linuron-induced alteration in genes encoding testosterone synthesis is likely a major factor in linuron-induced male reproductive toxicity.