Propionic acid (PA) is widely used in the food, agricultural, and pharmaceutical industries. Since the petrochemical PA is unsustainable, biological production of PA from renewable substrates is gaining attention. In this study, we engineered the strain Pseudomonas putida KT2440 to transform l-threonine to PA with only CO2 released as by-product. The cell factory was created by chromosomal incorporation of heterologous l-threonine deaminase, permease, and acyl-CoA thioesterase, deletion of branch pathways as well as overproduction of the endogenous branched-chain alpha-keto acid dehydrogenase complex. The final engineered strain could produce 399 mM PA from 400 mM l-threonine in a batch biotransformation process, with a molar yield of 99.8% under the optimized conditions in 48 h. The PA titer further reached to 50.3 g/L (679 mM) with a productivity of 0.6 g/L/h in a fed-batch conversion process. No obvious by-products, such as acetate and succinate, were detected in the broth, which would significantly facilitate downstream purification steps. Thus, this study offers an alternative route for biological production of PA.