Recently we demonstrated that the miRNA regulate human mesenchymal stem cells (hMSCs) differentiation. To determine the role of the miRNA pathway in hMSCs proliferation, Drosha and Dicer knockdown hMSCs were generated using a lentiviral based tetracycline inducible shRNA. hMSCs with reduced Drosha expression had a significantly reduced proliferation rate, while hMSCs with reduced Dicer expression displayed a proliferation rate similar to untransduced cells. Cell cycle analysis identified that unlike Dicer knockdown, Drosha knockdown hMSCs contained an increased number of G1 phase cells, with a reduced level of cells in S phase, compared to controls. ELISAs of hMSCs revealed decreased levels of pRB and stable levels of total RB with Drosha knockdown. Two key regulators of the G1/S phase transition, cyclin dependent kinase inhibitor 2A (p16) and cyclin dependent kinase inhibitor 2B (p15), were increased in Drosha knockdown cells but not in Dicer knockdown. Transcripts of 28S and 18S rRNA were significantly reduced in Drosha knockdown hMSCs, with no change in rRNA levels in Dicer knockdown hMSCs. 45S pre-rRNA transcripts were not significantly different in either knockdown model. The above results indicate that Drosha modifies hMSCs proliferation through a miRNA independent mechanism, potentially by regulating rRNA processing.