Yersinia pestis injects a set of virulent proteins into the cytosol of eukaryotic cells by a type III secretion system (T3SS). LcrG is a known negative regulator for secretion of Yersinia outer-membrane proteins (Yops) by blocking the secretion apparatus (Ysc) from the inner membrane. To further understand the effect of lcrG deletion on Y. pestis T3SS regulation, transcriptional profiles from the DeltalcrG mutant and wild-type Y. pestis strains were compared. The results showed that although the DeltalcrG mutant was markedly attenuated (600-fold increase of LD(50) in s.c. challenged BALB/c mice), transcriptions of almost all the type III genes were upregulated significantly in the DeltalcrG mutant. The immunoblotting analysis of YopM and LcrV demonstrated that their expressions were also increased in the DeltalcrG mutant in comparison to the wild-type strain. We speculate that, in addition to the negative regulation of the Yop secretion, LcrG could possibly play a negative regulatory role in the transcription of T3SS genes through indirect mechanisms. Furthermore, this report also revealed significant transcriptional changes in the genes encoding cell-envelope-related proteins and a virulence-related transcription factor RovA in the DeltalcrG mutant.