[Objective] We studied calcium-associated signal responses of Penicillium chrysogenum to phenoxyacetic acid. [Methods] The effects of different types of Ca2+ interference agents (Nifedipine, EGTA, Suramin and Neomycin sulfate) for penicillin V production were studied. Intracellular Ca2+ concentration was measured by Olympus System microscope and SpectraMax M2 Fluorescence Microplate Reader. [Results] Levels of intracellular Ca2+ in P. chrysogenum significantly increased (49.86% higher than the control) at 25 h after treatment with phenoxyacetic acid (POA), whereas significantly reduced (53.31% lower than the control) after treatment with neomycin sulfate as an intracellular calcium inhibitor. Results indicate that cells regulated intracellular calcium level though Ins(1,4,5)P3 signaling pathways to response POA stress. [Conclusion] Understanding the effect of Ca2+ signal transduction pathway how P. chrysogenum responses to POA stress provides guidance for industrial penicillin production.