As the most prominent member of the miR-17-92 cluster, miR-17-5p is well associated with tumorigenesis and cancer progression. It can exert both oncogenic and tumor-suppressive functions by inducing translational repression and/or mRNA decay. The complexity of the tissue-specific expression of the targeted transcripts seems to contribute to the differential functions of miR-17-5p in different types of cancers. In this study, we selected 12 reported miR-17-5p targeting genes with mRNA levels unaffected by miR-17-5p expression and analyzed their expression in 31 organ tissues in transgenic mice by real-time PCR. Surprisingly, miR-17-5p expressing transgenic mice showed a positive correlation in these tissues between miR-17-5p expression levels and the selected miR-17-5p targeted transcripts; with high expression of the miRNA in organs with high selected miRNA-targeted mRNA levels. In cancer cell lines, overexpression of 7 reported miR-17-5p targeted genes’ 3′-UTRs promoted miR-17-5p expression; meanwhile, transfection of 3′-UTRs with mutations had no significant effect. Moreover, an increase in AGO2 mRNA was associated with 3′-UTR expression as confirmed by real-time PCR. Hence, miR-17-5p regulation by these target genes might be an alternative mechanism to maintain miR-17-5p expression at tissue-specific levels.