The stereoisomerization of 2,5-disubstituted 1-zirconacyclopent-3-yne compounds, stable five-membered cycloalkynes, has been studied with regard to the mechanism. The bimetallic complex of 1,4-bis(trimethylsilyl)butatriene was synthesized and structurally characterized, although it seems unimportant for the stereoisomerization reactions. The isomerization of trans-1,1-bis(η 5 -cyclopentadienyl)-2,5-bis(trimethylsilyl)-1-zirconacyclopent-3-yne 2a into the cis-form in benzene-d 6 solution were observed using 1 H NMR spectroscopy at 50 °C in various concentrations. The reaction was first order with respect to trans-2a. This ruled out the possibility that a bimetallic complex was responsible for the isomerization. A kinetic isotope effect was observed (k H /k D = 1.8), suggesting that C–H activation is involved in the rate-determining step. A mechanism via hydrogen elimination from the complex of η 4 -π,π-coordination mode is proposed.