Salmonella is a major foodborne pathogen that can effectively replicate inside host macrophages to establish life-threatening systemic infections. Salmonella must utilize diverse nutrients for growth in nutrient-poor macrophages, but which nutrients are required for intracellular Salmonella growth is largely unknown. Here, we found that either acquisition from the host or de novo synthesis of a nonprotein amino acid, β-alanine, is critical for Salmonella replication inside macrophages. The concentration of β-alanine is decreased in Salmonella-infected macrophages, while the addition of exogenous β-alanine enhances Salmonella replication in macrophages, suggesting that Salmonella can uptake host-derived β-alanine for intracellular growth. Moreover, the expression of panD, the rate-limiting gene required for β-alanine synthesis in Salmonella, is upregulated when Salmonella enters macrophages. Mutation of panD impaired Salmonella replication in macrophages and colonization in the mouse liver and spleen, indicating that de novo synthesis of β-alanine is essential for intracellular Salmonella growth and systemic infection. Additionally, we revealed that β-alanine influences Salmonella intracellular replication and in vivo virulence partially by increasing expression of the zinc transporter genes znuABC, which in turn facilitates the uptake of the essential micronutrient zinc by Salmonella. Taken together, these findings highlight the important role of β-alanine in the intracellular replication and virulence of Salmonella, and panD is a promising target for controlling systemic Salmonella infection.