microRNAs (miRNA), a class of recently discovered small RNA consisted of 22 nucleotides, can regulate gene expression by binding to 3′-UTR of target mRNAs. miRNA can negatively regulate the expression of target mRNA at post-transcription and translation level. It is speculated that nearly 30% of the animal genes are regulated by miRNA. Along with the functional studies of miRNA, it is reported that the two important gene expression regulation factors——transcription factors and splicing factors have direct and/or indirect relationships with miRNA. The relationship between miRNA, transcription factors and splicing factors will provide a new insight of miRNA in understanding miRNA’s function and new clues for therapeutical application.