The Archean world-class Kirkland Lake Au deposit hosts characteristics of intrusion-related- and orogenic-type deposits and the origin of Au in this giant deposit has remained elusive. Here, the Li isotopic composition of hydrothermal alteration is combined with bulk compositions and petrographic observations to shed light on the genesis of the deposit. The Li isotopic composition was measured along a drill core cutting through comagmatic Timiskaming alkali tuff, Kirkland Lake mafic syenite and syenite porphyry, and the crosscutting fault zone (the ’04 Break). A combined increase in δ7Li, Li, Au, and major/trace elements with distance measured along the drill core in the lower alkali tuff was related with increased amounts of hydrothermal K feldspar and resulted from the injection of a magmatic-hydrothermal fluid, conferring δ7Li values of ~ +4‰ to the tuff. Kinetic isotopic fractionation resulted in a smooth decrease of δ7Li values from ~ +4‰ to ~ +2‰ down the Li concentration gradient and it is proposed that Au followed Li as colloidal particles along the interconnected network of fluid pathways. The upper syenite porphyry shows no variation of major/trace elements or mineralogy with drill hole distance but ranges in δ7Li values from ~ +5‰ to ~ +2‰, resulting from magmatic- or metamorphic-hydrothermal alteration and the recrystallization of white mica. A V-shape pattern is observed for δ7Li, Li and Au through the ’04 Break, with a low focused on the central veinlet and values increasing on both sides. Enhanced permeability at the fault centerline allowed the circulation of fluids, which leached Au and Li from the rock, triggered the recrystallization of white mica, and decreased the δ7Li values. Anomalously high δ7Li values of + 14.0‰ and + 16.4‰ were obtained for an auriferous (0.91 ppm Au) quartz-carbonate-pyrite veinlet, where the high δ7Li values are ascribed to the preference for 7Li of quartz. The Li isotopic values presented here are in agreement with previous studies suggesting that the auriferous hydrothermal alteration at Kirkland Lake was sourced from magmatic fluids and agree that metamorphic fluids and groundwaters were also involved in the hydrothermal alteration of the system.