Gender and prior knowledge may affect students' performance and motivation when simulations and games are used for learning. Accommodations should be made for students of different genders and with different levels of prior knowledge. A simulation digital game about the food chain concept geared for elementary school students was developed for this study. Participants were 114 fifth-grade students in Taiwan. A quantitative research method was used to examine the impact of gender and prior knowledge on students' learning performance and motivation. In-depth interviews were conducted to explore the reasons for these impacts. The results showed that the improvement in the learning performance of females was significantly better than that of males, especially in the low prior knowledge group. The reason may be that the males paid more attention to the progress and competition of the game missions, and tended to ignore content. Students with low prior knowledge were more likely to be affected by their environment and peers, which hindered attention. The constant clicking in the game distracted the attention of females with high prior knowledge.