The purpose of this embedded mixed-methods study was to a) determine whether song-based instruction effects foundational hematology knowledge acquisition as measured by hematology module summative exam scores for first-year physician assistant (PA) students and to b) explore student perceptions of song-based instruction on their medical knowledge acquisition. Information processing theory was used to understand mechanisms by which the song-based intervention might impact learning. The study included a sample of 100 first-year PA students enrolled in a foundational hematology module, 50 of whom received lecture-based instruction only, and 50 of whom received instruction which included the use of songs with content-specific lyrics. In the quantitative component of this study, a quasi-experimental, posttest only design was employed to evaluate for differences in assessment scores between cohorts. Quantitative subanalyses were performed to further evaluate the impact of the song-based intervention on assessment performance, including the impacts of familiar melody and musical experience. In the qualitative component of the study, interviews were conducted with students in the experimental cohort and data underwent thematic analysis to explore student perceptions of the song-based intervention. The quantitative analysis showed that there was no statistically significant difference (p = .400) in hematology module exam scores between the control (M = 95.36, SD = 3.713) and experimental (M = 96.00, SD = 3.854) cohorts. Quantitative subanalyses similarly resulted in no statistically significant differences in exam scores in the experimental cohort for test items covered in songs versus items not (p = .219), items taught by songs with familiar versus novel melodies (p = .894), and within the cohort based on musical ability (p = .811). Qualitative analysis resulted in three themes (Songs supported knowledge acquisition in multiple ways, Songs positively impacted the learning experience, and Songs facilitated reflection and connection) which suggested positive effects of the intervention that were not measured quantitatively. The findings from this study suggest that a song-based instructional approach has the potential to be an engaging, joyful, and impactful intervention to support the acquisition of foundational knowledge in health professions learners.