Heat shock response is a classical stress-induced regulatory system in bacteria, character-ized by extensive transcriptional reprogramming. To compare the impact of heat stress on the tran-scriptome and translatome in Escherichia coli, we conducted ribosome profiling in parallel with RNA-Seq to investigate the alterations in transcription and translation efficiency when E. coli cells were exposed to a mild heat stress (from 30 ?C to 45 ?C). While general changes in ribosome foot-prints correlate with the changes of mRNA transcripts upon heat stress, a number of genes show differential changes at the transcription and translation levels. Translation efficiency of a few genes that are related to environment stimulus response is up-regulated, and in contrast, some genes func-tioning in mRNA translation and amino acid biosynthesis are down-regulated at the translation level in response to heat stress. Moreover, our ribosome occupancy data suggest that in general ribosomes accumulate remarkably in the starting regions of ORFs upon heat stress. This study provides additional insights into bacterial gene expression in response to heat stress, and suggests the presence of stress-induced but yet-to-be characterized cellular regulatory mechanisms of gene expression at translation level.