Wnt/β-catenin signalling plays a prominent role in maintaining self-renewal and pluripotency of mouse embryonic stem cells (mESCs). microRNAs (miRNAs) have critical roles in maintaining pluripotency and directing reprogramming. To investigate the effect of GSK3 inhibitors on miRNA expression, we analysed the miRNA expression profile of J1 mESCs in the absence or presence of CHIR99021 (CHIR) or 6-bromoindirubin-3'-oxime (BIO) by small RNA deep-sequencing. The results demonstrate that CHIR and BIO decrease mature miRNAs of most miRNA species, 90.4% and 98.1% of the differentially expressed miRNAs in BIO and CHIR treated cells were downregulated respectively. CHIR and BIO treatment leads to a slight upregulation of the primary transcripts of the miR-302-367 cluster and miR-181 family of miRNAs, these miRNAs are activated by Wnt/β-catenin signalling. However, the precursor and mature form of the miR-302-367 cluster and miR-181 family of miRNAs are downregulated by CHIR, suggesting CHIR inhibits maturation of primary miRNA. Western blot analysis shows that BIO and CHIR treatment leads to a reduction of the RNase III enzyme Drosha in the nucleus. These data suggest that BIO and CHIR inhibit miRNA maturation by disturbing nuclear localisation of Drosha. Results also show that BIO and CHIR induce miR-211 expression in J1 mESCs.