Gliomas arising in the brainstem are rare tumours that are difficult to surgically resect, and the microRNAs (miRNAs) and signalling pathways associated with brainstem gliomas (BSGs) are largely unknown. To identify grade-associated miRNAs in BSGs, a microarray analysis of 10 low-grade and 15 high-grade BSGs was performed in this study. Differentially expressed miRNAs (DE-miRNAs) were identified, and the functional DE-miRNAs were selected. The potential target genes and enriched pathways were analysed, and a target gene-associated protein-protein interaction (PPI) network was generated. Grade-associated functional DE-miRNAs were confirmed by real-time quantitative PCR. First, 28 functional DE-miRNAs, including 13 upregulated miRNAs and 15 downregulated miRNAs, were identified. Second, 2546 target genes that were involved in BSG-related pathways, such as signalling pathways regulating the pluripotency of stem cells, the AMPK signalling pathway, the HIF-1 signalling pathway, the PI3K-Akt signalling pathway, the Wnt signalling pathway and the Hippo signalling pathway, were screened. Third, PHLPP2 and VEGFA were identified as hub genes in the PPI network. Last, we found that hsa-miR-34a-5p inhibits BSG cell invasion in vitro. In summary, using integrated bioinformatics analysis, we have identified the potential target genes and pathways of grade-associated functional DE-miRNAs in BSGs, which could improve the accuracy of prognostic evaluation. Furthermore, these hub genes and pathways could be therapeutic targets for the treatment of BSGs.