Low temperature scanning tunneling microscopy (LT-STM) has been applied to investigate the adsorption of acetylene (C2H2) on the rutile TiO2(110) surface at 77 K. Through in situ exposing and ex situ annealing experiments we unambiguously demonstrate that C2H2 molecules adsorb weakly on top of the Ti5c ions. Moreover, our data clearly reveal that the oxygen vacancy (Ov) cannot directly adsorb C2H2 molecules but can activate its next-nearest Ti5c ions which register as a binding site for C2H2 molecules with enhanced stability. Further increasing the C2H2 coverage leads to the stepwise formation of c-(4 × 2) and c-(2 × 2) ordered structures before final saturation at around 0.68 monolayer. The saturated film is characterized by dominating C2H2 dimers and other minority linear clusters whose intermolecular distances are slightly larger than the intervals between Ti5c ions yet significantly shorter than the C2H2 dimers in the gas phase. We propose that such clustered C2H2 may serve as potential precursors for the polymerization reactions on metal oxide surfaces.