Alterations in the intracellular levels of calcium are a common response to cell death stimuli in animals and fungi and, particularly, in the Neurospora crassa response to staurosporine. We highlight the importance of the extracellular availability of Ca²⁺ for this response. Limitation of the ion in the culture medium further sensitizes cells to the drug and results in increased accumulation of reactive oxygen species (ROS). Conversely, an approximately 30-fold excess of external Ca²⁺ leads to increased drug tolerance and lower ROS generation. In line with this, distinct staurosporine-induced cytosolic Ca²⁺ signaling profiles were observed in the absence or presence of excessive external Ca²⁺. High-throughput RNA sequencing revealed that different concentrations of extracellular Ca²⁺ define distinct transcriptional programs. Our transcriptional profiling also pointed to two putative novel Ca²⁺-binding proteins, encoded by the NCU08524 and NCU06607 genes, and provides a reference dataset for future investigations on the role of Ca²⁺ in fungal biology.