is among the best known medicinal basidiomycetes due to its production of many pharmacologically active compounds. To study the regulatory networks involved in its growth and development, we analyzed the relationship between reactive oxygen species (ROS) and Ca signaling in the regulation of hyphal branching and ganoderic acid (GA) biosynthesis after Cu treatment. Our results revealed that Cu treatment decreased the distance between hyphal branches and increased the GA content and the intracellular levels of ROS and Ca Further research revealed that the Cu-induced changes in hyphal branch distance, GA content, and cytosolic Ca level were dependent on increases in cytosolic ROS. Our results also showed that increased cytosolic Ca could reduce cytosolic ROS by activating antioxidases and modulating Cu accumulation, resulting in feedback to adjust hyphal growth and GA biosynthesis. These results indicated that cytosolic ROS and Ca levels exert important cross talk in the regulation of hyphal growth and GA biosynthesis induced by Cu Taken together, our results provide a reference for analyzing the interactions among different signal transduction pathways with regard to the regulation of growth and development in other filamentous fungi., which is known as an important medicinal basidiomycete, is gradually becoming a model organism for studying environmental regulation and metabolism. In this study, we analyzed the relationship between reactive oxygen species (ROS) and Ca signaling in the regulation of hyphal branching and ganoderic acid (GA) biosynthesis under Cu stress. The results revealed that the Cu-induced changes in the hyphal branch distance, GA content, and cytosolic Ca level were dependent on increases in cytosolic ROS. Furthermore, the results indicated that increased cytosolic Ca could reduce cytosolic ROS levels by activating antioxidases and modulating Cu accumulation. The results in this paper indicate that there was important cross talk between cytosolic ROS and Ca levels in the regulation of hyphal growth and GA biosynthesis induced by Cu.