The spatiotemporal regulation of Ca²⁺ channels at the plasma membrane in response to extracellular signals is critical for development, stress response and reproduction, but is poorly understood. During flowering-plant reproduction, pollen tubes grow directionally to the ovule, which is guided by ovule-derived signals and dependent on Ca²⁺ dynamics. However, it is unknown how ovular signals are integrated with cytosolic Ca²⁺ dynamics in the pollen tube. Here, we show that MILDEW RESISTANCE LOCUS O 5 (MLO5), MLO9 and MLO15 are required for pollen tube responses to ovular signals in Arabidopsis thaliana. Phenotypically distinct from the ovule-bypass phenotype of previously identified mutants, mlo5 mlo9 double-mutant and mlo5 mlo9 mlo15 triple-mutant pollen tubes twist and pile up after sensing the ovular cues. Molecular studies reveal that MLO5 and MLO9 selectively recruit Ca²⁺ channel CNGC18-containing vesicles to the plasma membrane through the R-SNARE proteins VAMP721 and VAMP722 in trans mode. This study identifies members of the conserved seven transmembrane MLO family (expressed in the pollen tube) as tethering factors for Ca²⁺ channels, reveals a novel mechanism of molecular integration of extracellular ovular cues and selective exocytosis, and sheds light on the general regulation of MLO proteins in cell responses to environmental stimuli.