Contamination by and persistence of pathogenic bacteria in ready-to-eat produce have emerged as significant food safety and public health concerns. Viable produceborne pathogens cope with several stresses (e.g., temperature fluctuations and lowtemperature storage) during production and storage of the commodities. In this study, we investigated the impact of transient cold shock on Escherichia coli O157:H7 (EcO157) cells in a produce matrix (romaine lettuce leaf lysate). EcO157 cells were exposed to 25°C for 1 h, 4°C for 1 h, and 4°C for 10 min in lettuce lysate. The expression of selected genes coding for virulence, stress response, and heat and cold shock proteins was quantified by real-time quantitative reverse transcription PCR assay. Treated EcO157 cells adhered to MAC-T mammalian cells were enumerated by in vitro bioassay. Expression of the Shiga toxin 1 gene (stx1a) was upregulated significantly (P < 0.05) upon cold shock treatments, but virulence genes related to EcO157 attachment (eaeA, lpfA, and hcpA) were down-regulated. Two key members of the cold shock regulon, cold shock protein (cspA) and gyrA, were significantly induced (P < 0.05) at the refrigeration temperature (4°C). Significant upregulation of an SOS response gene, recA, was also observed. E. coli heat shock regulon member grpE was induced, but a universal stress protein (uspA) was downregulated at the refrigeration temperatures in lettuce lysate. The adhesion assay revealed a temperature-dependent reduction in the attachment of cold-shocked EcO157 cells. The results of the current study indicate a reduction in the attachment of cold-shocked EcO157 to epithelial cells and higher levels of Shiga toxin gene expression at the molecular level.