The prestressed RC-UHPC composite box girder with high tensile strength and corrosion-resistant ultra-high performance concrete (UHPC) as the web and bottom plate and reinforced concrete (RC) as the top plate was proposed, and a specimen was fabricated. The flexural test was carried out to analyse the crack development pattern and damage mode of the specimen; the finite element software was used to simulate the test, and the calculation accuracy was verified by comparing the finite element calculation results with the testing data, on the basis of which 19 finite element analysis models were established to analyse the influence of various parameters on the cracking moment; relevant codes and existing literature were used to investigate the cracking moment of the prestressed RC-UHPC composite box girder. The results show that: the first crack appeared when the specimen was loaded to about 33.3% of ultimate load; the cracking moment was influenced by the prestressing bar tension factor, the prestressing bar reinforcement ratio and the tensile strength of UHPC; the cracking moment calculation method based on the conversion section and the crack width calculation formulas in DBJ 43/T 325—2017 were used to calculate the cracking moment and crack width of the prestressed RC-UHPC composite box girder, respectively, and the calculated values agreed well with the testing and FE results.