Transforming growth factorβ1 (TGFβ1) is known to be intimately involved in many cellular processes. To explore the mechanism of TGFβ1 in these processes, the non-chimeric hammerhead ribozyme and U1 snRNA chimeric ribozyme against TGFβ1 were designed to down-regulate TGFβ1 expression. The activity of non-chimeric ribozyme and U1 snRNA chimeric ribozyme against TGFβ1 in vitro and in activated hepatic stellate cells (HSCs) was detected. Cleavage reactions of both ribozymes in vitro demonstrated that non-chimeric ribozyme possessed better cleavage activity in vitro than U1 snRNA chimeric ribozyme. The further study showed U1 snRNA chimeric ribozyme inhibited TGFβ1 expression more efficiently than non-chimeric ribozyme in transfected HSC cells. So it indicates that the U1 snRNA chimeric ribozyme provides an alternative approach for the research on the precise mechanism of TGFβ1 in many cellular processes and a potential therapeutic candidate for TGFβ1-related diseases.