The activity of Fischer-Tropsch synthesis (FTS) on metal-based nanocatalysts can be greatly promoted by the support of reducible oxides, while the role of support remains elusive. Herein, by varying the reduction condition to regulate the TiOx overlayer on Ru nanocatalysts, the reactivity of Ru/TiO2 nanocatalysts can be differentially modulated. The activity in FTS shows a volcano-like trend with increasing reduction temperature from 200 to 600 °C. Such a variation of activity is characterized to be related to the activation of CO on the TiOx overlayer at Ru/TiO2 interfaces. Further theoretical calculations suggest that the formation of reduced TiOx occurs facilely on the Ru surface, and it involves in the catalytic mechanism of FTS to facilitate the CO bond cleavage kinetically. This study provides a deep insight on the mechanism of TiOx overlayer in FTS, and offers an effective approach to tuning catalytic reactivity of metal nanocatalysts on reducible oxides.