Prestressed cold-formed U-section steel-concrete composite beams (CUCCBs) are formed by connecting cold-formed U-section steel to concrete through shear connectors and applying prestressing. Such composite beams have the advantages of easy construction, strong adaptability, high bearing capacity, high bending stiffness, and good global and local stability. However, there are less studies on prestressed CUCCBs under positive and negative bending moments compared with I-shaped steel-concrete composite beam. In this paper, four-point bending tests were conducted on prestressed CUCCBs with different configurations (with/without prestressing, prestressing locations) and loading conditions (positive/negative moments, simply supported/continuous spans) to investigate their bending performance. The results showed that the concrete slab in prestressed CUCCB subjected to negative bending moment cracked relatively early, with a cracking load of about 13 % of the ultimate load. The prestressed continuous CUCCB had the best bending performance and could be promoted to practical applications. Under the ultimate state, the continuous composite beam was damaged by partial concrete crushing and yielding of tensile steel plate, with small mid-span deformation of about 10 mm. In addition, a simple but effective nonlinear analysis model was also established by developing appropriate code, and the accuracy of the model was verified through experimental results.