Anticipation Guides (AGs) help learners to activate prior knowledge before an instructional unit. As a pre-learning strategy, AGs motivate learners to explore learning materials by challenging, activating, or corroborating their prior knowledge and predictions about a subject. While AGs have mostly been used in reading instruction, in this study, we evaluated the extent to which their use can influence visual attention distribution and learning in a multimedia environment. Eye tracking data from 17 participants randomly assigned to a treatment (with AG) or control group (without AG) demonstrated a significant difference in visual attention distribution but not on learning outcomes. Learners who used the AG exhibited larger numbers of transitions between text and images on the screen. The relevance of this study is two-fold: a) it contributes to the literature on anticipation guides as a learning strategy to activate prior knowledge; and b) it contributes to the literature on eye tracking methodology to support research on allocation of visual attention distribution in a multimedia learning environment.