Glucose phosphate isomerase (GPI) involves in the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate in glucose pathways. Because glucose metabolism is crucial for the proliferation and differentiation of embryonic stem and germ cells, reducing GPI expression may affect the characteristic features of these cells. MicroRNAs (miRNAs) have been shown to regulate genes. In the present study, we investigated the regulation of chicken GPI by its predicted miRNAs. We determined the expression patterns of seven GPI 3′-untranslated region (3′UTR)-targeting miRNAs, including the gga-miR-302 cluster, gga-miR-106, gga-miR-17-5p, and gga-miR-20 cluster in chicken primordial germ cells (PGCs), compared with GPI mRNA. Among the miRNAs, gga-miR-302b, gga-miR-302d, and gga-miR-17-5p were expressed at lower levels than GPI mRNA. The remaining four miRNAs—gga-miR-302c, gga-miR-106, gga-miR-20a, and gga-miR-20b—were expressed at higher levels than the expression of GPI mRNA. Next, we cotransfected four candidate miRNAs—gga-miR-302b, gga-miR-106, gga-miR-17-5p, and gga-miR-20a—with GPI 3′UTR into 293FT cells by dual fluorescence reporter assay. Overexpression of gga-miR-302b and gga-miR-17-5p miRNAs in 293FT cells significantly downregulated GPI expression, whereas the other two miRNAs had no effect. Then, knockdown and overexpression of these four candidate miRNAs were performed by RNA interference assay to regulate GPI in PGCs. In the RNA interference assay, the expression of GPI was greatly regulated by gga-miR-302b and gga-miR-17-5p. Finally, we examined the effects of GPI regulation on PGC proliferation and migration. Our results suggested that the regulation of GPI by gga-miR-302b and gga-miR-17-5p affected PGCs proliferation. However, regulation of GPI using these two miRNAs did not affect the migration of PGCs into embryonic gonads.