Cronobacter sakazakii is a food-borne pathogen that can thrive in various environments, including the human body. The human body’s physiological temperature exceeds that of the environment (22–30 °C), necessitating adaptations to heat stress during this transition. Managing heat stress is crucial when transitioning from the environment to the human body. In this study, we explored the effect of human body temperature on the growth of planktonic C. sakazakii, as well as its acid resistance, osmotic stress resistance, autoaggregation, and cell surface hydrophobicity. Our study demonstrated that human body temperature facilitated the growth, acid resistance, and osmotic resistance of C. sakazakii, compared to 28 °C. The relationship between human body temperature and phenotypes was studied by comparing gene expression at human and environmental temperatures (37 to 28 °C) using high-throughput sequencing. The results revealed up-regulation in the expression of 626 genes, including genes involved in arginine and proline metabolism, carbon fixation pathways, and nitrogen metabolism. Further analysis showed that human body temperature is essential for the environmental stress resistance of C. sakazakii. It boosts denitrification, betaine transport, and universal stress proteins, supporting membrane integrity and osmoprotectant transport. This study enhances our understanding of the strategies employed by C. sakazakii during its adaptation to the human body.