This research was aimed to analyse students’ emotions caused by the character’s realism design in the dimension of arousal and its’ effect on emotions in learning. Thus, 2D female virtual agents with four different realism appearance namely realistic, semi-realistic, stylized and cartoon-like were designed and developed. Thus, quasi experimental design was used to answer the research questions derived from this study. The data obtained from the experiment was analysed using ANOVA, post hoc and bootstrap mediation analysis. A number of 600 Electrical Engineering students were chosen from seven polytechnics in Malaysia as respondents in the experiment. From the research it was found that cartoon-like design obtained the lowest mean score arousal followed by stylized, realistic and semi-realistic designs. However, cartoon-like design scored highest mean score for emotions in learning followed by semi-realistic, realistic and stylized designs. Therefore, cartoon-like agent is the best agent among others in inducing positive emotions in learning. In addition, the mediation analysis shows that arousal has mediating effects on relation between different realism designs and emotions in learning. In conclusion, this study recommends cartoon-like agent as the best suitable design for 2D virtual agent followed by semi-realistic, realistic and stylized designs subsequently. Finally, the findings of this study can be a useful guideline for multimedia designers in determining the ideal 2D virtual agent appearance to elicit maximum impact of it in promoting positive emotions in multimedia learning environment.