Relatively few studies have been focused so far on magnesium–isotope fractionation during plant growth, element uptake from soil, root-to-leaves transport and during chlorophylls biosynthesis. In this work, maize and garden cress were hydroponically grown in identical conditions in order to examine if the carbon fixation pathway (C4, C3, respectively) might have impact on Mg-isotope fractionation in chlorophyll-a. The pigment was purified from plants extracts by preparative reversed phase chromatography, and its identity was confirmed by high-resolution mass spectrometry. The green parts of plants and chlorophyll-a fractions were acid-digested and submitted to ion chromatography coupled through desolvation system to multiple collector inductively coupled plasma-mass spectrometry. Clear preference for heavy Mg-isotopes was found in maize green parts (?26Mgplant-nutrient 0.65, 0.74 for two biological replicates, respectively) and in chlorophyll-a (?26Mgchlorophyll-plant 1.51, 2.19). In garden cress, heavy isotopes were depleted in green parts (?26Mgplant-nutrient (−0.87)–(−0.92)) and the preference for heavy isotopes in chlorophyll-a was less marked relative to maize (?26Mgchlorophyll-plant 0.55–0.52). The observed effect might be ascribed to overall higher production of energy in form of adenosine triphosphate (ATP), required for carbon fixation in C4 compared to C3, which could reduce kinetic barrier and make equilibrium fractionation prevailing during magnesium incorporation to protoporphyrin ring.