Spatial variation in chemical defense of plants can be caused by genetic, biotic, and abiotic factors. For example, many plants exhibit a latitudinal cline in chemical defense, potentially due to latitudinal variation in abiotic environmental factors such as the light regime during the growing season. In the worldwide distributed Plantago lanceolata, the levels of deterrent iridoid glycosides (IGs), aucubin and catalpol, vary geographically, including latitudinally. To examine whether latitudinal variation in photoperiod can explain part of this geographic variation, plants from the Netherlands and Finland were exposed to two different photoperiods, simulating the Dutch (middle European) and Finnish (northern European) light period during the growing season. The experiment showed that although most variation in IG content was genetic, plants from both Dutch and Finnish origin produce relatively more catalpol under a northern European than under a middle European photoperiod. Our results confirm that latitudinal effects on photoperiod can contribute to geographic variation in plant defense chemistry, which should be considered when studying latitudinal clines in plant-enemy interactions.