Objective：To investigate the expression of long non-coding RNA（lncRNA）and mRNA in the genital tubercle of hypospadiac rats induced by di-n-butyl phthalate（DBP）. Methods：We placed male and female rats with a ratio of 1∶1 in the same cage and then checked if there was white glue frosting vaginal suppository in the next day to determine the female rats mating successfully，confirmed this day as the first day of pregnancy. By giving rats 800 mg/kg DBP dissolved in 1 mL corn oil in the 13 to 18 days of pregnancy to establish model of hypospadias，the pregnant rats in the control group were given only 1 mL/d corn oil. Fetal rats were taken out from pregnant rats for 19 days of pregnancy. The meatus of urethra was observed with an anatomical microscope to find if male fetal rats developed hypospadias. Then，we removed the genital tubercle of male fetal rats and extracted total RNA in it. RNA sequencing library was constructed and then sequenced with sequencing machine. The sequencing result of lncRNA was analyzed and screened，and function prediction was performed. Finally，we chose eight lncRNAs randomly to validate their level of expression by qRT-PCR. Results：The sequencing results showed that compared with the control group，the hypospadias group had 598 differential expression mRNA，427 differential expression lncRNAs. qRT-PCR results were consistent with the results of sequencing. In the biological process，cell component and molecular function，the most differential expressions of mRNA were related to erythrocyte development，extracellular exosome and haptoglobin binding. The most mRNAs were related to the signaling pathways of viral myocarditis. LncRNAs function prediction showed that the most lncRNAs played in combination of growth hormone releasing hormone receptor；The most lncRNAs were related to the signaling pathways of staphylococcus aureus infection. Conclusion：Compared with normal male rats，the male rats of hypospadias induced by DBP have changed the level of expression of lncRNA and mRNA in the genital tubercle significantly. LncRNA may involved in the formation process of hypospadias and interfered development of genital tubercle by interacting with mRNA.