We report the controlled synthesis and structures of two isomeric gold nanoclusters, whose compositions are determined to be Au23(C≡CBut)15 (denoted as Au23-1 and Au23-2) by single-crystal X-ray diffraction and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. This is the first time isomerism is discovered in alkynyl-protected gold nanoclusters. The metal-to-ligand ratios in these two clusters are different from known Aun(SR)m systems and have not been observed in the Aux(C≡CPh)y family. This pair of isomers exhibits different optical properties, although they have similar structures and identical components. For both Au23 clusters, time-dependent density functional theory calculations revealed the frontier orbitals highest occupied molecular orbital (HOMO)-1, HOMO, and lowest unoccupied molecular orbital (LUMO) are mainly constructed from the Au15 kernel and V-shaped alkynyl-gold motifs. The HOMO → LUMO transition of Au23-1 is optically forbidden, whereas it is allowed in Au23-2. It is also found that Au23-2 cluster can be transformed to Au23-1 spontaneously under ambient conditions. This work offers further insight into the synthesis and isomerism of all-alkynyl-protected gold nanoclusters and will stimulate more investigation of isomeric metal nanoclusters.