MiRNAs are known as small, non-coding and single stranded RNAs which can regulate cell proliferation, differentiation, apoptosis, and involve in the development of sperm. Meanwhile, DUSP4, PDK4 and FKBP1B were also found to participate in testis development and spermatogenesis. In this study, online software and luciferase reporter gene system were used to predict and verify the target relationship between miR-122 and DUSP4/PDK4, miR-449a and FKBP1B. Thereafter, comparison analysis was done on miR-122, miR-449a, DUSP4, PDK4 and FKBP1B mRNA expression levels in different bull testes tissues (1-day, 12-months-old and 24-months-old). In addition, genes mRNA and protein levels were detected after bull testicular Sertoli cells were transfected with miR-122 and miR-449a. Meanwhile, DUSP4, PDK4 and FKBP1B overexpression experiments were conducted. Eventually, MTT assay was performed to observe the cells proliferation. The results showed that miR-122 and miR-449a were highly-expressed in testis tissues (p<0.01). The expression levels of miR-122 and miR-449a in the 24-month-old testes were higher than those in the neonatal and 12-month-old groups during the testicular maturation process, however, PDK4, DUSP4 and FKBP1B expression levels were decreased (p<0.01). Furthermore, the luciferase in miR-122/449a co-transfected with pmiR-RB-REPORT-DUSP4-WT or pmiR-RB-REPORT-PDK4/FKBP1B-WT group was significantly lower than pmiR-RB-REPORT-DUSP4-mut or PDK4/FKBP1B-mut and negative control groups. Meanwhile, PDK4, DUSP4 and FKBP1B expression levels were down-regulated due to miR-122 and miR-449a overexpression (p<0.01). Moreover, cells proliferation ability was activated after DUSP4, PDK4 and FKBP1B overexpression (p<0.01). Finally, MTT assay results demonstrated that miR-122 or miR-449a could promote bull testicular Sertoli cells proliferation. Collectively, these data suggested that miR-122/PDK4/DUSP4 and miR-449a/FKBP1B pathways could play a vital role in bull testis development.