<p>Alkynes play a crucial role in chemical synthesis, bio-imaging, and drug design. Despite their significance, the intermolecular interactions involving alkynes have been largely unexplored. In this work, we unveil the previously overlooked alkyne-π interaction by comparing two zirconocene metal-organic cage compounds. The distinct stacking geometry in the single-crystal structures, coupling with the changes in C ≡ C vibrational signals, confirms the alkyne-π interaction as a genuine intermolecular interaction. Combining with computational studies, we reveal that alkyne-π interactions exert a substantial influence on the spectroscopic properties, despite being energetically less potent than π-π interactions. Our findings extend beyond theoretical implications. A comprehensive survey of the Cambridge Crystallographic Data Centre (CCDC) database corroborates the occurrence of alkyne-π interactions across hundreds of crystal structures, which provides a missing piece for fundamentally rationalizing their properties. Meanwhile, the changing C ≡ C vibrational signals, under alkyne-π interactions, may provide strategies for improving bio-imaging resolutions. It could also serve as a signature for desired alkyne-containing supramolecular structures. These results highlight the potential of alkyne-π interactions in designing functional materials for advanced applications in chemistry and biology.</p><p></p>