Abstract: In order to investigate the mechanical properties of steel and concrete composite girders with circle tubular up-flange in negative moment region, five simply supported composite girders under negative moment were tested by static loading. According to the characteristics of test girders at the strength limit state, the formulas for the ultimate moment capacity in pure bending and the ultimate shear strength in pure shear for circle tubular up-flange composite girders under negative moment were developed based on the simplified plastic theory, which were then compared with the experimental results to evaluate the calculation precision. The moment-shear (M-V) interaction from EC4, GB 50917-2013, ASCE specifications and the formulas proposed by Liang et al. as well as the corresponding predictive values of ultimate strength were compared, and an applicable form of M-V interaction was found to well estimate the M-V strength interaction for circle tubular up-flange composite girders in negative moment region. The results indicate that the test girders show excellent stability and ductility during loading. Four typical failure modes, including local shear buckling, lateral buckling of the lower flange, web crippling at the end of test girders and lateral flexural-torsional buckling of the steel beam, take place together in the last damage state. After the concrete slab crack in tension, the calculated elastic shear strain distribution without consideration of the contribution from the concrete slab agrees well with the measured shear strain curves. Furthermore, the M-V interaction should be considered in calculating the ultimate strength of circle tubular up-flange composite girders in the negative moment region due to the influence of high-shear and high-moment. The M-V strength interaction from GB 50917-2013, with the average accuracy of 96% compared with the test values, provides the best combination of accuracy and safety of prediction. Based on the application of formulas for the ultimate moment capacity in pure bending and the ultimate shear strength in pure shear proposed by this paper, it is suggested that the M-V strength interaction according to GB 50917-2013 should be used to calculate the ultimate strength of circle tubular up-flange composite girders under negative moment.