Ganoderma lucidum has drawn worldwide interest with regard to its secondary metabolism and pharmaceutical activity. However, the development of such research has been limited because of a lack of basic biological knowledge. Nicotinamide adenine dinucleotide phosphate oxidases (Nox) have recently been highlighted because of the many important biological roles in plants and animals; however, the exact functions of Nox are still not fully understood in fungi. In this study, we identified two Nox isoforms (NoxA and NoxB) and a regulator, NoxR. RNA interference was used, and silencing of the Nox isoforms and NoxR expression indicated a central role for these genes in hyphal branching, fruiting body development, reactive oxygen species (ROS) generation, ROS resistance and ganoderic acid biosynthesis regulation. Further mechanistic investigation revealed that Nox-generated ROS elevated cytosolic Ca2+ levels by activating a plasma membrane Ca2+ influx pathway, thereby inducing the Ca2+ signal pathway to regulate ganoderic acid biosynthesis and hyphal branching. Importantly, our results highlight the Nox functions in signal crosstalk between ROS and Ca2+, and these findings provide an excellent opportunity to identify the potential pathway linking ROS networks to calcium signalling in fungi and suggest that plants, animals and fungi share a conserved signal-crosstalk mechanism.