Pedagogical agents were found to enhance learning but studies on the emotional effects of such agents are still missing. While first results show that pedagogical agents with an emotionally positive design might especially foster learning, these findings might depend on the gender of the agent and the learner. This study investigated whether emotional expressions performed by an on-screen instructor were able to increase learning outcomes while considering differences the gender of the agent and the learner. In a 2 (neutral vs. enthusiastic expressions) × 2 (female vs. male agent) between-subject design with additional consideration of the gender of the learner, data of 129 participants was collected. Results revealed that the manipulation of enthusiasm lead to higher perceptions of positive emotions. In addition, a pedagogical agent who performed enthusiastic expressions led to a higher retention but not transfer performance. In terms of the gender of the agent and the learner, male learners retained knowledge better when they watched the agent performing enthusiastic expression irrespective of the persona gender. Female learners, however, retained knowledge only better when a female agent performed enthusiastic expressions. Results are discussed in the light of the positivity principle, model-observer similarity hypotheses and current theories on social cues in multimedia learning.