Supported sulfide vanadium-based catalyst in dehydrogenation of light alkanes is becoming a promise to replace the commercial used Pt- and Cr-based catalysts, due to the lower cost and toxicity. Nonetheless, the nature of active sites and reaction mechanism for dehydrogenation of isobutane over sulfide vanadium-based catalysts are still ambiguous. In this study, a series of sulfide V-K/γ-Al2O3 catalysts were prepared, presenting diverse distribution of isolated, oligomeric and polymeric vanadium species on catalyst surface. The oligomeric species were demonstrated to be the active dehydrogenation sites, and the TOFoligo showed a closely correlation with the isobutane conversion. Besides, the dehydrogenation reaction mechanism was studied by in-situ DRIFTS. The results showed that isobutane would first dissociative adsorbed on catalyst surface, and the stable intermediates of vanadium-isobutenyl were detected. Furthermore, the deposited coke rather than sulfur loss was found to play the crucial role in deactivation. The vast majority of deposited coke existed as polyaromatic species and the other slight ones presented as aliphatic form. This study enriches the fundamental explorations in active sites and reaction mechanism of sulfide V-K/γ-Al2O3 catalysts during isobutane dehydrogenation, and provides an in-depth strategy to improve the activity and stability of vanadium-based dehydrogenation catalysts.