The positive regulatory machinery in the microRNA (miRNA) processing pathway is relatively well characterized, but negative regulation of the pathway is largely unknown. Here we show that a complex of nuclear factor 90 (NF90) and NF45 proteins functions as a negative regulator in miRNA biogenesis. Primary miRNA (pri-miRNA) processing into precursor miRNA (pre-miRNA) was inhibited by overexpression of the NF90 and NF45 proteins, and considerable amounts of pri-miRNAs accumulated in cells coexpressing NF90 and NF45. Treatment of cells overexpressing NF90 and NF45 with an RNA polymerase II inhibitor, alpha-amanitin, did not reduce the amounts of pri-miRNAs, suggesting that the accumulation of pri-miRNAs is not due to transcriptional activation. In addition, the NF90 and NF45 complex was not found to interact with the Microprocessor complex, which is a processing factor of pri-miRNAs, but was found to bind endogenous pri-miRNAs. NF90-NF45 exhibited higher binding activity for pri-let-7a than pri-miR-21. Of note, depletion of NF90 caused a reduction of pri-let-7a and an increase of mature let-7a miRNA, which has a potent antiproliferative activity, and caused growth suppression of transformed cells. These findings suggest that the association of the NF90-NF45 complex with pri-miRNAs impairs access of the Microprocessor complex to the pri-miRNAs, resulting in a reduction of mature miRNA production.