Environmental factors applied in animal research facilities need to be regulated accurately not only for the welfare of laboratory animals but also for the reliability of the experimental results. However, the effect of lighting environment and its characteristics on animals has been poorly investigated. The aim of our experiment was to study the potential preference of C57BL/6J mice between two different light colour temperatures by using a 1 h preference test. During the test, mice were allowed to choose between a cage illuminated with warm fluorescent light (2500 K) and another with cool fluorescent light (4000 K). After assessing the amount of time spent by mice in each cage, we found that they presented a significant preference for the warm lightilluminated cage. Even though mice continued to visit both cages during the test, a preference was established over time, especially during the second 30 min interval. However, mice engaged in higher levels of locomotor activity in the cool light-illuminated cage compared to the other as a result of their exposure to light of different colour temperatures. These findings emphasize the importance of properly regulating the various aspects of lighting in order to guarantee better housing conditions for laboratory animals and reproducibility and reliability of experimentation.