Activation of the AKT (serine-threonine kinase) pathway is a common feature in glioblastoma cells. Downstream factors of the AKT pathway are involved in cell proliferation, apoptosis, cellular migration and angiogenesis. Micro-RNAs (miRNAs) are highly conserved small non-coding RNAs that block targeted mRNA expression at the post-transcriptional level. The aim of this study was to investigate the role of the AKT pathway in regulating miRNA. The changes of miRNA expression profile in human glioblastome U251 cells after AKT small interfering RNA transfection were examined by a microarray, and confirmed by Northern blotting. Down-regulation of AKT expression by siRNA decreased the activity of AKT pathway in U251 cells. Interruption of AKT pathway suppressed the expression of NF-kappaB and c-Myc, furthermore, the expression of a set of miRNAs was also changed after AKT siRNA transfection. There are putative binding sites of NF-kappaB and c-Myc in the promoters of several up-regulated miRNAs, indicating these transcription factors may also be involved in the regulation of miRNA expression, thus affecting the activity of AKT pathway in tumorigenesis. We provide new components of the regulatory function of AKT pathway to better understand the regulatory network mediated by downstream transcription factors. The understanding of the regulatory function of AKT pathway is crucial in tailored therapy of gliomas.