Hypoxia (H) in solid tumors contributes to decreased immunosurveillance via Kv1.3 channel downregulation and inhibition of T cell function. However, the mechanisms responsible for Kv1.3 downregulation are not understood. We tested the hypothesis that chronic H (CH) reduces Kv1.3 surface expression via alterations in membrane trafficking. Jurkat T cells were maintained either in normoxia (21% O2) or H (1% O2) for 24h and Kv1.3 surface expression was quantified by in‐cell western blot analysis and flow cytometry using a specific Kv1.3 antibody against an extracellular epitope. CH decreased Kv1.3 surface expression in T cells by 25%. To study the involvement of endocytosis, endosome formation was blocked with Bafilomycin A1 (BAF). Comparable inhibition of Kv1.3 surface expression in CH was observed in BAF (27±4%) and control (ctr) (25±3%) cells. On the contrary, inhibition of the Golgi apparatus by Brefeldin A (BFE) prevented Kv1.3 surface expression reduction in CH (ctr 25±2% and BFE −4±3%). Furthermore, blockade of clathrin‐coated vesicle formation in the Golgi by dynasore (DYN), a selective dynamin inhibitor, prevented Kv1.3 surface expression decrease in CH (ctr 23±0.5% and DYN 0.93±1%). Our data suggest that CH disrupts forward trafficking of Kv1.3 protein from the Golgi to the plasma membrane thus contributing to the decrease in Kv1.3 surface expression in T lymphocytes. (NIH 2R01CA095286)