Many signal perception mechanisms are connected to Ca-based second messenger signaling to modulate specific cellular responses. The well-characterized plant hormone auxin elicits a very rapid Ca signal. However, the cellular targets of auxin-induced Ca are largely unknown. Here, we screened a biologically annotated chemical library for inhibitors of auxin-induced Ca entry in plant cell suspensions to better understand the molecular mechanism of auxin-induced Ca and to explore the physiological relevance of Ca in auxin signal transduction. Using this approach, we defined a set of diverse, small molecules that interfere with auxin-induced Ca entry. Based on annotated biological activities of the hit molecules, we found that auxin-induced Ca signaling is, among others, highly sensitive to disruption of membrane proton gradients and the mammalian Ca channel inhibitor bepridil. Whereas protonophores nonselectively inhibited auxin-induced and osmotic stress-induced Ca signals, bepridil specifically inhibited auxin-induced Ca We found evidence that bepridil severely alters vacuolar morphology and antagonized auxin-induced vacuolar remodeling. Further exploration of this plant-tailored collection of inhibitors will lead to a better understanding of auxin-induced Ca entry and its relevance for auxin responses.