Dendritic cells (DCs) are the sentinels of immunity ideally equipped with unique properties to transport antigens from the periphery to lymphoid tissues and to present them to T cells. Toll like receptor 4 (TLR4, one of the pattern recognition receptors) expressed on DC surface responds to lipopolysaccharides (LPS), elements of the outer wall of Gram-negative bacteria. Upon TLR4 ligation, DCs start the maturation reprogramming which initial steps include stimulation of antigen endocytosis and enhanced reactive oxygen species (ROS) production. Both phagosomal pH and ROS production depend on NADPH oxidase (NOX2), which activity requires continuous pH and charge compensation. The aim of this study was to investigate the mechanisms involved in pH regulation and charge adjustment in DCs upon TLR4 stimulation. In patch-clamp whole cell experiments, we showed the functional expression of Hv1 proton channels in mouse bone marrow-derived DCs. Whole cell outwardly rectifying currents measured with TMASO₃ pipette and bath solutions were activated upon depolarization. The threshold voltage of activation (V_threshold) was dependent on the pH gradient between pipette and bath solution and was close to the empirically predicted V_threshold for the Hv1 currents. LPS had bimodal effects on Hv1 channels: acute LPS treatment (1 mg/ml) increased Hv1 channel activity, whereas the 24 hours LPS incubation significantly inhibited Hv1 currents, aligned with reduction in Hv1 mRNA expression levels determined by RT-PCR and decline in ROS production as measured by DCFDA fluorescence in FACS. Our investigation reveals involvement of Hv1 in pH regulation and charge compensation during TLR4 ligation in DCs.