Shiga toxins (Stxs), some of the most important virulence factors in enterohemorrhagic Escherichia coli (EHEC) O157:H7, are known to be induced and released by various environmental cues, such as DNA damage responses and stress-inducing chemicals. In order to investigate the possible effects of growth media on Stxs expression, we analyzed the growth kinetics and expression of Stxs (Stx1 and 2) in cells grown in Luria-Bertani (LB) and E. coli (EC) media, which are widely used for EHEC O157:H7. Through direct plating and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), it was revealed that, when EHEC O157:H7 was grown in EC medium, the total bacterial count was reduced significantly and the stx1 transcription was greatly increased during the stationary growth phase than that in LB. Here we report that bile salts and lactose, which are the two only components in EC medium that are absent in LB, function as negative and positive regulatory signals, respectively, for the transcription of both stx1 and stx2. Indeed, stx transcription was significantly increased (~5.7 and ~21.8 fold for stx1 and stx2, respectively; p < 0.05) in an EC medium lacking bile salts when compared to the normal EC. In contrast, EHEC O157:H7 grown in an EC medium lacking lactose did significantly decrease these transcriptions (~93.5 and ~4.3 fold for stx1 and stx2, respectively; p < 0.05). Consistently, stx transcription was drastically increased in an LB medium supplemented with lactose, implying that lactose might be an environmental trigger for the expression of Stxs.