Abstract Moonlight exerts profound ecological, behavioural and physiological effects on animals. However, lunar cycles are characterised by complex changes in the illuminance and timing of illumination, making it challenging to re‐create and manipulate moonlight cycles in the laboratory using artificial lights. As a result, ecological experiments on the effects of moonlight cycles are uncommon, and existing studies often oversimplify the re‐creation of moonlight. This limitation extends to experimental studies of the effects of light pollution on nocturnal animals, which often fail to adequately represent natural nocturnal light. To address the lack of open‐source solutions for re‐creating and manipulating moonlight cycles, we developed the software‐hardware system MoonShine. This has two components: (1) MoonShineR, an R package with additional R scripts, which predicts moonlight ground illuminance (in lux) at defined intervals, for a specified location and time range; (2) MoonShineP, a Python program running on a Raspberry Pi computer, which uses the illuminance values from MoonShineR to gradually dim and brighten a diffused array of individually addressable LEDs, allowing realistic natural light regimes to be re‐created in a laboratory environment. MoonShine includes multiple features to re‐create and manipulate light cycles. It supports colour‐shifting of the LED light (by adjustment of RGBW intensity ratios) to approximate the spectrum of natural moonlight, and to mimic habitat‐specific conditions or certain types of light pollution. We tested the accuracy of MoonShineR's moonlight illuminance predictions by comparison to field radiometer measurements at equatorial and temperate latitude sites. We demonstrated the accuracy of MoonShineP's moonlight re‐creation, by comparing its measured LED illuminances to the intended values and its measured LED spectrum against natural moonlight. MoonShine allows researchers to re‐create a range of natural nocturnal lighting scenarios in the laboratory. It can be used to re‐create full natural moonlight cycles with a relatively realistic spectral composition, generate manipulated moonlight schedules, or simulate light pollution. Furthermore, the moonlight illuminance predicted by MoonShineR is useful for field ecologists who require moonlight as a quantitative model predictor. Finally, to provide laboratory‐housed animals with full diurnal light cycles, MoonShine allows researchers to re‐create natural twilight and sunlight regimes.