Abstract Calcium, an ubiquitous second messenger, plays an essential and versatile role in cellular signaling. The diverse function of calcium signals is achieved by an excess of calcium sensors. Plants possess large numbers of calcium sensors, most of which have not been functionally characterized. To identify physiologically relevant calcium sensors in a specific cell type, we conducted a genome‐wide functional survey in pollen tubes, for which spatiotemporal calcium signals are well‐characterized and required for polarized tip growth. Pollen‐specific members of calmodulin (CaM), CaM‐like (CML), calcium‐dependent protein kinase (CDPK) and calcineurin B‐like protein (CBL) families were tagged with green fluorescence protein (GFP) and their localization patterns and overexpression phenotypes were characterized in tobacco pollen tubes. We found that several fusion proteins showed distinct overexpression phenotypes and subcellular localization patterns. CDPK24‐GFP was localized to the vegetative nucleus and the generative cell/sperms. CDPK32‐GFP caused severe growth depolarization. CBL2‐GFP and CBL3‐GFP exhibited dynamic patterns of subcellular localization, including several endomembrane compartments, the apical plasma membrane (PM), and cytoskeleton‐like structures in pollen tubes. Their overexpression also inhibited pollen tube elongation and induced growth depolarization. These putative calcium sensors are excellent candidates for the calcium sensors responsible for the regulation of calcium homeostasis and calcium‐dependent tip growth and growth oscillation in pollen tubes.