In this study, Al2O3 thin films are deposited on p-type silicon using spatial atomic layer deposition with trimethylaluminum and H2O. The films are annealed in atmosphere (ATM), forming gas (FG) and nitrogen (N2) at temperatures of 300–750 °C. Effects of annealing gas ambient and temperature on structural, electrical, and passivation properties of Al2O3/Si are systematically investigated. The experimental results show that the ATM annealing leads to an interfacial silicon oxide more than two times thicker than that of the samples annealed in FG and N2. The ratio of tetrahedral AlO4 to octahedral AlO6 can be correlated to the negative fixed oxide charge density (Qf) near the interface of Al2O3/Si. The 600 °C ATM-annealed sample has the highest Qf of 3.23 × 1012 cm−1 and thus gives the best field effect passivation. The sample annealed in FG at 450 °C has the lowest interface trap density (Dit) of 3.98 × 1011 eV−1 cm−2, indicating that the hydrogen is more effective than oxygen for chemical passivation. Overall, the FG-annealed sample has the highest lifetime of 933.8 μs, showing that chemical passivation is the primary consideration. The ATM annealing requires higher temperatures (~600 °C) to reach the optimal passivation, whereas the FG and N2 annealing temperature is limited to 450 °C to avoid dehydrogenation. Finally, for passivated emitter and rear contact cell fabrication, the cell with FG annealing has the best conversion efficiency of 21.43%, which is 0.17 and 0.54 percentage point higher than that of the cells with ATM and N2 annealing, respectively.