Radio frequency (RF) has found its potential in ensuring the safety of packaged powdered infant formula milk (PIFM). PIFM was processed by RF at different temperature-holding time treatments (70 °C-115 min, 75 °C-50 min, 80 °C-25 min, 85 °C-10 min, and 90 °C-5 min) to obtain 5-log reduction of Cronobacter sakazakii, and its physicochemical characteristics were investigated. Various RF treatments possessed insignificant effects on color difference (ΔE < 1.5), solubility (>99%), crystallinity (∼5%), surface chemical composition (>30% fat, 15%–20% protein, and 45%–50% lactose), and microstructure of PIFM. Lower temperature-longer holding time treatment as 70 °C-115 min caused 51% reduction in water activity and 71% reduction in moisture content, thereby considerably increasing the glass transition temperature of PIFM from 60.3 °C to 78.4 °C. Protein secondary structures were preserved; however, 75 °C-50 min and 80 °C-25 min treatments significantly (P < 0.05) decreased the α-helix structure. All RF heating conditions significantly (P < 0.05) altered the concentration of volatile organic compounds in PIFM and increased the relative abundance of lipid oxidation substances such as (E)-2-heptenal, 2-heptanone, and n-nonanal. PIFM can be treated at 90 °C for 5 min for better quality and processing efficiency.