Salmonellae are often present as immobilized cells in food products in the form of micro-colonies. Despite this, most research into Salmonella physiology has been performed with bacteria grown as planktonic cultures. In the current study, we compared the transcriptome of planktonic and immobilized Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) subjected to 30 min of heat stress at 45 °C. The expression of 538 genes was differently regulated between the two conditions after 30 min. Heat stress of an immobilized culture induced expression of flagella and virulence genes compared to the non-heat stressed immobilized bacteria. Immobilized heat stressed S. Typhimurium was more invasive in HeLa cells than the non-heat stressed controls, whereas the heat stress caused planktonic bacteria to be less invasive. The decrease in invasion of heat stressed planktonic cultures returned to non-heat stressed levels 30 min post treatment, whereas the increased invasion of HeLa cells of the heat stressed immobilized cultures still remained 30 min after the heat stress was terminated. This study shows that immobilized bacteria respond to heat stress differently than planktonic bacteria.