The male hypemethylated (MHM) region, associated with sex-specific DNA methylation and histone acetylation, encodes non-coding RNAs that are expressed exclusively on Z chromosome in female chickens. The function of the MHM non-coding RNAs is unclear yet. In order to analyze the regulation effect of MHM on mRNA expression of sex-dependent genes, exogenous plasmid containing chicken MHM (cMHM) was constructed and injected into left testes of 13-week-old roosters. Comb color of the roosters in the pEGFP-N1-cMHM group became much paler, an indication of change in sex hormones, as compared with that of the negative control groups at the 10 days after injection. Real-time PCR was used to determine mRNA expression of sex-dependent genes in testes tissue. Transcription of ER alpha gene was significantly up-regulated in the pEGFP-N1-cMHM group compared with that of the pEGFP-N1 and normal saline groups (P < 0.01), while expression of DMRT1 mRNA declined severely in the pEGFP-N1-cMHM group compared with that of normal saline groups (P < 0.05). Nevertheless, expression of other candidate genes, such as AMH, FOXL2, SOX9, and P450arom did not change after injection of exogenous cMHM. In conclusion, down-regulation of DMRT1 and up-regulation of ER alpha in rooster testes treated with exogenous pEGFP-N1-cMHM plasmid might indicate a novel cMHM non-coding RNA-mediated mechanism in avian sex differentiation.