Na+/Ca2+ exchange is important to cell physiology and metabolism, but its role in the secondary metabolite biosynthesis by fungi is yet unclear. In this work, in static liquid cultures of Ganoderma lucidum, which is an efficient process for hyper-production of anti-tumor ganoderic acids (GAs), it was interestingly found that Na+ addition could enhance the GAs production, but K+ did not. Further investigation by intracellular Ca2+ imaging and using a calcineurin inhibitor (i.e., cyclosporin A) revealed that addition of Na+ led to the influx of Ca2+ from culture broth to the cells and calcineurin signals were also triggered. Addition of 100 mM Na+ at the beginning of the static liquid cultivation, in which the addition dosage and timing were optimized, resulted in 2.8-fold improvement of total GAs production. Quantitative gene transcription analysis indicated that the expression levels of the genes of Ca2+ sensors and GA biosynthesis were upregulated with Na+ induction while downregulated by using the calcineurin inhibitor, implying that higher GA production might result from higher expression of those genes. This work not only provided a simple and efficient induction strategy by Na+ addition for the improved GAs production but also suggested the regulation mechanism of Na+ on the GA biosynthesis through calcineurin signaling transduction. Biotechnol. Bioeng. 2013; 110: 1913–1923. © 2013 Wiley Periodicals, Inc.