To investigate the possible molecular mechanism of low concentration plasticizer mono-n-butyl phthalate (MBP)-induced juvenile Sertoli cells (SCs) proliferation, we evaluated global alterations of miRNA and mRNA expression in rat SCs treated with 0.1 mM MBP. Microarray analysis revealed that miR-3584-5p and miR-301b-3p were up-regulated and their common target gene Dexamethasone-induced Ras-related protein 1 (Rasd1) was down-regulated. Further work suggested that SCs proliferation induced by low concentration MBP in vitro might be mediated by Rasd1 regulating ERK1/2 signaling pathway. The present study is first to investigate the effect of low-dose MBP on SCs proliferation and may enhance our understanding on the modes of action of low concentration MBP on male reproductive system. We hope the results will contribute to explain the causes of precocious puberty and testicular tumors induced by exogenous chemicals.