Owing to the high metal binding affinity of metallothionein (MT), newly synthesized MT was speculated to attenuate the activity of metal-responsive element (MRE)-binding transcriptional factor 1 (MTF-1) by removing Zn from the activated MTF-1. To investigate the potential role of MT in the inactivation of MTF-1, we examined the transcriptional levels of reporter and endogenous MRE-dependent genes using mouse embryonic fibroblasts (MEFs) established from MT-knockout (KO) and wild-type (WT) mice. The activation of MTF-1 by the Cd exposure of MT-KO MEFs was sustained for 12 h, whereas that of MT-WT MEFs showed the rapid attenuation. Consequently, MT was found to negatively regulate MTF-1 activity, which can control the expression of MT itself.