Soil salinization is a significant factor that severely limits the production of high-quality sugar beet in China. However, little is known about the physiological and molecular regulatory mechanism of sugar beet in response to salt stress. In this study, salt-tolerant (AK3018) and salt-sensitive varieties (IM1162) were screened from 50 sugar beet cultivars, and transcriptome analysis identified 3281, 2614, 1930, and 4866 differentially expressed genes (DEGs) in the AK_L_C-VS-AK_L_S, AK_R_C-VS-AK_R_S, IM_L_C-VS-IM_L_S, and IM_R_C-VS-IM_R_S groups, respectively. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that DEGs responsive to salt stress were significantly enriched in multiple metabolic pathways, including proline and betalain biosynthesis, antioxidant enzyme activity, chlorophyll biosynthesis, and ion transmembrane transport. Additionally, the contents of proline, betaine, and soluble sugar and the activities of catalase and ascorbate peroxidase were significantly increased in sugar beet under salt stress. AK3018 had higher chlorophyll content, photosystem II activity, and more K+ and less Na+ in leaf than IM1162 under salt stress. These results indicate that sugar beet can accumulate osmoregulatory substances, maintain the reactive oxygen species balance, improve the photosynthetic system, and reconstruct ion homeostasis in response to salt stress. The results provide a deeper understanding of the physiological and molecular mechanisms of sugar beet in response to salt stress and provide a large number of candidate genes for molecular salt tolerance breeding in sugar beet.