Mpv17-like protein (M-LP) is a protein that has been suggested to be involved in the metabolism of reactive oxygen species. The two M-LP isoforms in mouse, M-LP(S) and M-LP(L), are generated by the alternative usage of promoters. M-LP(S) is expressed exclusively in kidneys after the age of 6 weeks, whereas M-LP(L) is expressed ubiquitously. To elucidate the molecular basis of M-LP(S) expression, we searched for cis-regulatory elements in the promoter region of M-LP(S) and identified heat shock element half-sites as positive elements and a Tramtrack 69K (Ttk 69K) binding site as a negative element. Furthermore, we isolated a novel transcription repressor, Rhit (regulator of heat-induced transcription), that binds to the Ttk 69K binding site within the M-LP(S) promoter by DNA affinity chromatography and confirmed its participation in the transcriptional regulation of M-LP(S) by RNA interference (RNAi). Sequence analysis revealed that Rhit contains a KRAB (Krüppel-associated box) domain and a DNA-binding domain composed of eight C(2)H(2)-type zinc fingers. Interestingly, exposure to heat shock stress resulted in the upregulation of M-LP(S) expression concurrent with the downregulation of Rhit expression. Moreover, the age-dependent expression of M-LP(S) was inversely correlated with that of Rhit. These observations strongly suggest that Rhit acts as a repressor in the heat-induced and age-dependent transcriptional regulation of M-LP(S).