The response of Oenococcus oeni SD-2a to short-term acid stress (3 h) was detected by RNA sequencing (RNA-seq). The transcriptome data were analyzed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Besides, some physiological indicators related to the stress response were also measured. Based on the obtained results, a schematic diagram illustrating the stress response mechanism was drawn. It turned out that under acid stress condition, the bacterial cells synthesized more ATP to pump H⁺ out of them, and the ATPase activity was significantly increased. A suitable pH could maintain an appropriate enzymatic reaction environment for life activities. The cell membrane fluidity was decreased by adjusting the contents of total unsaturated fatty acids and total cyclic fatty acids. In conclusion, the intracellular energy production was greatly increased, and some energy dissipation responses favorable for resistance to acid stress were significantly increased, while some unrelated energy dissipation responses were inhibited. Furthermore, in order to deal with the damage caused by acid stress, the expression levels of DNA-repairing genes and chaperone protein genes were greatly increased. Our findings provide insights into the regulatory network associated with acid stress responses in O. oeni.