In 2010, the Member States of the E.U. established a directive concerning the protection of animals, stipulating the limitations of the use of animals for scientific or education purposes only in cases where all measures aimed at avoiding such use have failed. One of the solutions, within the educational framework, where the observed results during experimentation are expected, is the use of simulation with serious games (SG). The use of SG has demonstrated their relevance in general college education, even though not all studies agree on the game elements of SG that promote academic outcomes. However, in pharmacology education, few studies have looked at the effect of serious games on these various academic outcomes, and in particular at the game elements that can improve them. In this context, the aim of this work was to have a better understanding of the role of a serious game in the education of pharmacology students by studying the effect of environment (virtual/non-virtual) and the ability to interact with it (active/passive) on cognitive (acquisition of knowledge, cognitive load), behavioral (engagement) and affective (motivation, emotions) outcomes. In total, 46 first year undergraduate students participated in a pharmacology lesson through SG (involving a virtual laboratory) or a course slideshow, both in active or passive modality. Results have shown that the virtual environment in active mode can lead to better behavioral (engagement) and affective (enjoyment, intrinsic motivation, etc.) outcomes, but to similar cognitive outcomes, in comparison with slideshows. These results and proposals for future work are discussed.