Yersinia enterocolitica is a major foodborne pathogen causing yersiniosis, a significant zoonotic infection. Its unique cold tolerance makes it a potential threat to the safety of cold chain food. The RNA-binding protein Hfq is known to be involved in bacterial stress regulation, but its role in the cold tolerance of Y. enterocolitica remains unclear. Based on gene knockout and complementation, this study revealed that deleting the hfq gene disrupted the exponential growth phase of Y. enterocolitica under low-temperature conditions, leading to a biphasic growth phenomenon. Additionally, through the construction of point mutants, it was found that the critical amino acid sites for cold-tolerance regulation of Hfq in Y. enterocolitica are located on the distal and proximal surfaces. Further studies found that the biphasic growth process of the hfq deletion mutant was affected by the initial bacterial concentration and exogenous fatty acids. RT-qPCR results showed that Hfq regulation may affect the synthesis of branched-chain amino acids, and TCA cycle-related genes were significantly up-regulated during the second exponential growth. Studies based on milk and meat have shown that Hfq can promote the low-temperature growth of Y. enterocolitica in the food matrix. Our study provides evidence that Hfq-dependent regulation of energy metabolism is critical for cold tolerance in Y. enterocolitica. This study highlights the importance of Hfq in regulating cold tolerance in Y. enterocolitica and discusses its potential regulatory mechanism.