Integrating simple molecular units into organometallic architectures has attracted significant research interest due to its wide applications in functional materials and devices. Despite recent progresses, it remains challenging to manipulate metal-molecule coordination with single molecule precision. Here, we create organometallic molecular junctions via terminal alkynes and measure their conductance in situ at the single molecule level using the scanning tunneling microscope-break junction (STM-BJ) technique. The strong Ag–C σ bonding between alkynes and Ag atoms drives the formation of linear organometallic −C≡C–Ag–C≡C– wires anchored between Ag or Au electrodes. Moreover, the conductance of the organometallic junctions can be precisely modulated through modifying the structures of the alkyne ligands. Our results suggest a versatile bottom-up approach for building organometallic nanostructures and studying their chemical and conducting properties.