The immunological synapse (IS), a highly organized structure that forms at the point of contact between a T cell and an APC, is essential for the proper development of signaling events, including the Ca2+ response. Kv1.3 channels control Ca2+ homeostasis in human T cells and move into the IS upon Ag presentation. However, the process involved in channel accumulation in the IS and the functional implications of this localization are not yet known. Here we define the movement of Kv1.3 into the IS and study whether Kv1.3 localization into the IS influences Ca2+ signaling in Jurkat T cells. Crosslinking of the channel protein with an extracellular Ab limits Kv1.3 mobility and accumulation at the IS. Moreover, Kv1.3 recruitment to the IS does not involve the transport of newly synthesized channels and it does not occur through recycling of membrane channels. Kv1.3 localization in the IS modulates the Ca2+ response. Blockade of Kv1.3 movement into the IS by crosslinking significantly increases the amplitude of the Ca2+ response triggered by anti-CD3/anti-CD28-coated beads, which induce the formation of the IS. On the contrary, the Ca2+ response induced by TCR stimulation without the formation of the IS with soluble anti-CD3/anti-CD28 Abs is unaltered. The results presented herein indicate that, upon Ag presentation, membrane-incorporated Kv1.3 channels move along the plasma membrane to localize in the IS. This localization is important to control the amplitude of the Ca2+ response, and disruption of this process can account for alterations of downstream Ca2+-dependent signaling events.