Compared with simply supported beams, continuous steel-concrete composite beams have many advantages such as higher span/depth ratio, less deflection, and higher fundamental frequency of vibration due to its higher stiffness. However, in negative bending regions near interior supports, tension in concrete is unfavorable and a complicated issue, which deserves a special study. In this paper, a mechanics model based on elastic theory was established to investigate the stiffness of composite beams in negative bending regions by considering slips at the steel beam-concrete slab interface and concrete-reinforcement interface. In order to validate this approach, a test of three composite beams with profiled sheeting under negative bending was conducted. Meanwhile, a three-dimensional nonlinear finite element (FE) analysis was conducted to investigate the general behavior of the tested specimens. In addition, a comparative analysis between results derived from the analytical model, laboratory test, and FE analysis was performed. The results show that slip always exists for composite beams under negative bending even with complete shear connection (full composite action) between the steel and concrete components. The slip effect results in an additional curvature of beam bending and reduces the section stiffness by 10-20% compared with that of a beam without any slip in serviceability condition. This reduction should be considered in designing process especially for cantilever beams. Formulae under other loading cases and boundary conditions were also proposed. The results can serve as a basis for further study on stiffness of continuous steel-concrete composite beams and can directly be used for the deflection calculation of cantilever beams.