Polysemy in graph-related practices is the phenomenon that a single graph can sustain different meanings assigned to it. Considerable research has been done on polysemy in graph-related practices in school science in which graphs are rather used as scientific tools. However, graphs in science textbooks are also used rather pedagogically to illustrate domain-specific textbook content and less empirical work has been done in this respect. The aim of this study is therefore to better understand polysemy in the domain-specific pedagogical use of graphs in science textbooks. From socio-cultural and cultural-historical perspectives, we perceive polysemy as irreducible to either the meaning-making (semiotic) resources provided by the graph or its readers who assign meaning to it. Departing from this framework, we simultaneously investigated: (a) the meanings 44 pre-university biology students assigned to the Cartesian plane of a graph that is commonly used as a pedagogical tool in Dutch high school biology textbooks (an electrocardiogram); (b) the semiotic resources provided by this graph; and (c) the educational practices of which it is supposedly a part according to the actions constituted by the textbooks that were to be conducted by students. Drawing on this case, we show polysemy in the pedagogical use of graphs in science textbooks. In turn, we show how this polysemy can be explained dialectically as the result of both the meaning-making resources provided by the textbooks and the graph-related practices in which students supposedly engaged by using their textbooks. The educational implications of these findings are discussed.