The hammerhead ribozyme is often used for gene regulations in a cell. One of the major differences between in vitro and in cell conditions is the molecular crowding. However, the influence of crowding conditions on the ribozyme activity is still unclear. Here, we investigated the activity of the hammerhead ribozyme under molecular crowding condition by ethylene glycol (EG), poly (ethylene glycol) (PEG), dextran, and Ficoll. These cosolutes enhanced the hammerhead ribozyme activity by 2∼7-times, and larger-sized PEGs exhibited the greater activity. More importantly, the rate constant at 1 mM Mg2+ and 37°C increased about 1000-times and 500-times upon the addition of 20 wt% PEG8000 and PEG200, respectively. Additionally, the ribozyme still retained the cleavage activity even at 50°C in the presence of PEG8000. Our results suggest that the ribozyme activity can be enhanced by the addition of the crowding reagent even at a low magnesium ion concentration and even at a high temperature.