Due to its structural efficiency, durability, and cost-effectiveness, ultra-high performance concrete was utilized to build the first highway overpass bridge in China. The bridge was made of prestressed ultra-high performance concrete box girders of four continuous spans of 30 m each. As the original design of such bridge was observed to be somewhat conservative, its cross-sectional dimensions, in the form of the box girder wall thicknesses were optimized in this research to lower the material cost in future bridge construction. Then, a full-scale simply supported ultra-high performance concrete box girder of 30 m span, incorporating the new box girder wall thicknesses, was fabricated and then tested under static loading to obtain research data to justify the revised design. The loading system was designed to examine the flexural behavior of the girder using two concentrated loads symmetrically located at the mid-span. Experimental results show that the optimized girder has a favorable ductile behavior and excellent flexural strength, which can meet the design requirements for serviceability and ultimate limit states. A finite element model of the tested girder was developed, using ABAQUS software, and then was verified using the experimental findings. A parametric study was then conducted to investigate the influence of key parameters on the structural response, namely, the reinforcement ratio, the number of the prestressing wires, and the web thickness. Recommendations on minimum and maximum compressive strength and tensile property of ultra-high performance concrete were proposed. Also, a simplified calculation method of prestressed ultra-high performance concrete box girder was developed based on a verified strain and stress diagrams for cross-sectional analysis. The proposed methodology can be used in future practice with confidence.