The rotating and thermal effects on sealing performance were analyzed and the calculation model of axial lateral straight-through labyrinth seal was established, in order to explore the sealing mechanism of high-speed gearbox with mixed media of lubricating oil and ideal gas. The influences of structure deformation and flow field on the sealing performance were also studied, which were induced by the rotating and thermal effects. The results reveal that the rotating and thermal effects can reduce the sealing gap. Nevertheless, the thermal-effect-induced expansive deformation exceeded the rotating-effect-induced centrifugal deformation by one order of magnitude. In addition, the rotation speed had a threshold value (4 000 r/min). When the rotor rotation speed exceeded this threshold, the leakage loss of labyrinth seal decreased significantly. Compared with the condition without rotation, the leakage loss decreased 18.5% under the rotation speed of 10 000 r/min. At the same time, the leakage loss increased linearly with the increment of lubricating oil temperature and the decrease of oil viscosity. When the oil temperature was at 140 °C, the leakage loss increased 58.6% in comparison with that at 40 °C. The flow filed caused by rotating and thermal effects is the major factor affecting the leakage performance of gearbox sealing system, while the structure deformation is the secondary factor.