Reactive oxygen species (ROS)-induced oxidative stress has been associated with diseases such as amyotrophic lateral sclerosis, stroke, and cancer. While the effect of ROS on mitochondria and endoplasmic reticulum (ER) has been well documented, its consequence on the Golgi apparatus is less well understood. In this study, we characterized the Golgi structure and function in HeLa cells after exposure to hydrogen peroxide (H2O2), a reagent commonly used to introduce ROS to cells. Treatment of cells with 1 mM H2O2 for 10 min resulted in the degradation of Arl1 and dissociation of GRIP domain-containing proteins Golgin-97 and Golgin-245 from the trans-Golgi. This effect could be rescued by treatment of cells with a ROS scavenger N-acetyl cysteine or protease inhibitors. Structurally, H2O2 treatment reduced the number of cisternal membranes per Golgi stack, suggesting a loss of trans-Golgi cisternae. Functionally, H2O2 treatment inhibited both anterograde and retrograde protein transport, consistent with the loss of membrane tethers on the trans-Golgi cisternae. This study revealed membrane tethers at the trans-Golgi as novel specific targets of ROS in cells.