Because U-shaped steel beams filled with concrete have the advantages of reduced construction period and superior structural performance, they have been widely used as structural elements. In this study, the flexural behavior of newly developed bolt-connected U-shaped steel and concrete composite beams under a negative bending moment was experimentally evaluated. The main test variables were bolt spacing in bottom steel plates and types of U-shaped steel sections. Test results showed that the flexural behavior of the composite beams is determined by the plate-buckling mode of bolt-connected bottom steel plates. Composite beams with a greater bolt spacing than that in the standard AISI S100-16 experienced early plate buckling of the bottom steel plates, resulting in reduction of flexural stiffness and maximum flexural strength. Thus, the buckling mode of bolt-connected bottom plates should be considered for bolt-connected U-shaped composite beams under a negative bending moment. The longitudinal lips of U-shaped steel sections can increase the flexural performance of composite beams due to anchorage performance of the steel sections with lips.