BACKGROUND: There is growing interest in the therapeutic potential of music or light in different human disorders. AIMS: This study aimed to evaluate the effects of music as well as darkness on FDG uptake in 4T1 tumor-bearing BALB/c mice using a PET scan. METHODS: The music, darkness, and music plus darkness groups were subjected to either song or darkness and their combination, respectively, 30 min before the radiopharmaceutical injection until the end of the experiments. The control group was imaged in silence under ambient conditions. RESULTS: Our results revealed that music did not significantly alter the range of tumor SUVmean, but showed a slight increase in brain SUVmean (18.2%) and about 100% increase in brain percentage of injected dose per gram (%ID/g) in CONCLUSION: Our results suggest that music and environmental factors may influence FDG uptake in small-animal PET imaging, and provide important insights into the reliability of FDG-PET imaging for music intervention research and may aid researchers in investigating the effects of music on brain changes and tissue metabolism.