Thermo-mechanical fatigue (TMF) is one of the most detrimental failures of critical engine components and greatly limits their service life. In this study, the out-of-phase TMF (OP-TMF) behavior in Al-Si-Cu 319 cast alloys microalloyed with Mo was systematically investigated under various strain amplitudes ranging from 0.1–0.6% and temperature cycling at 60–300 °C and compared with the base 319 alloy free of Mo. Cyclic stress softening occurred in both experimental alloys when applying the TMF loading, resulting from the coarsening of θ’-Al2Cu precipitates. However, the softening rate of the Mo-containing alloy was lower than that of the base 319 alloy because of its lower θ’-Al2Cu precipitate coarsening rate per cycle. The Mo-containing alloy exhibited a longer TMF lifetime than the base alloy at the same strain amplitude. Microalloying 319 alloy with Mo enhanced the TMF resistance mainly by slowing the coarsening of θ’-Al2Cu precipitates and providing supplementary strengthening from thermally stable Mo-containing α-dispersoids distributed in the Al matrix. The energy-based model was successfully applied for predicting the TMF lifetime with a low life predictor factor, which agreed well with the experimentally measured fatigue cycles.