Salmonella is a widespread zoonotic enteropathogen that causes gastroenteritis and fatal typhoidal disease in mammals. During systemic infection of mice, Salmonella enterica serovar Typhimurium resides and replicates in macrophages within the "Salmonella-containing vacuole" (SCV). It is surprising that the substrates and metabolic pathways necessary for growth of S. Typhimurium within the SCV of macrophages have not been identified yet. To determine whether S. Typhimurium utilized sugars within the SCV, we constructed a series of S. Typhimurium mutants that lacked genes involved in sugar transport and catabolism and tested them for replication in mice and macrophages. These mutants included a mutant with a mutation in the pfkAB-encoded phosphofructokinase, which catalyzes a key committing step in glycolysis. We discovered that a pfkAB mutant is severely attenuated for replication and survival within RAW 264.7 macrophages. We also show that disruption of the phosphoenolpyruvate:carbohydrate phosphotransferase system by deletion of the ptsHI and crr genes reduces S. Typhimurium replication within RAW 264.7 macrophages. We discovered that mutants unable to catabolize glucose due to deletion of ptsHI, crr, and glk or deletion of ptsG, manXYZ, and glk showed reduced replication within RAW 264.7 macrophages. This study proves that S. Typhimurium requires glycolysis for infection of mice and macrophages and that transport of glucose is required for replication within macrophages.