Updated FTP location. Ca2+ is a second messenger that regulates a variety of cellular responses in fungi. However, the mechanism behind how Ca2+ signals are transmitted within the cell as they regulate downstream targets remains unclear. We used a fluorescent Ca2+ biosensor to visualize the changes in Ca2+ concentration in the hyphae of the filamentous fungus Aspergillus nidulans in response to thermal stress via infrared laser point irradiation, and then we analyzed the characteristics of the periodic Ca2+ influx in the hyphae. Stress-responsive Ca2+ pulses promoted the reorganization of the actin cytoskeleton and exocytosis, and they controlled the resumption, redirection, or branching of hyphal growth. To clarify how calcium signals are transmitted to downstream targets, we focused on calmodulin (CaM) and calcium/CaM-dependent protein kinases (CaMKs; CmkA, CmkB, and CmkC) by clarifying their localization and studying their interacting proteins by GFP-trap and MS analysis. Analysis of proteins that co-precipitated with CaM or CamKs showed that the calcium signaling pathway is involved in actin cytoskeleton rearrangement, vesicle transport, cell wall synthesis, and the cell wall integrity (CWI) pathway. Our approach enabled us to visualize the actual conduction of Ca2+ in hyphae in response to stress, and it clarified the downstream targets of CaM and CaMKs, thereby connecting the series of events, from input to output that are involved in calcium signaling.