Ca2+ is an essential ion in the control of pollen germination and tube growth. However, the control of pollen tube development by Ca2+ signaling and its interactions with cytoskeletal components, energy-providing pathways, and cell-expansion machinery remain elusive. Here, we used nifedipine (Nif) to study Ca2+ functions in differential protein expression and other cellular processes in Pinus bungeana pollen tube growth. Proteomics analysis indicated that 50 proteins showed differential expression with varying doses of Nif. Thirty-four of these were homologous to previously reported proteins and were classified into different functional categories closely related to tip-growth machinery. Blocking the L-type Ca2+ channel with Nif in the pollen tube membrane induced several early alterations within a short time, including a reduction of extracellular Ca2+ influx and a subsequently dramatic decrease in cytosolic free Ca2+ concentration ([Ca2+]), concomitant with ultrastructural abnormalities and changes in the abundance of proteins involved in energy production and signaling. Secondary alterations included actin filament depolymerization, disrupted patterns of endocytosis/exocytosis, and cell wall remodeling, along with changes in the proteins involved in these processes. These results suggested that extracellular Ca2+ ` influx was necessary for the maintenance of the typical tip-focused [Ca2+] gradient in the P. bungeana pollen tube, and that reduced adenosine triphosphate production (ATP), depolymerization of the cytoskeleton, and abnormal endocytosis/exocytosis, together with enhanced rigidity of cell walls, were responsible for the growth arrest observed in pollen tubes treated with Nif.