The water-well recovered from a Late Pre-Pottery Neolithic B (PPNB) level in Tell Seker al-Aheimar, Northeast Syria, represents the oldest well thus far known in Upper Mesopotamia. It demonstrates that the construction of water-wells was a wide-spread practice among the PPNB communities across Cyprus, the Mediterranean coast, and now a far inland region of Upper Mesopotamia. This article provides detailed data on the water-well’s excavation and its stratigraphy, morphology, spatial positioning in the settlement, dating, and associated artefacts. An important implication of these data is that the construction and use of this well involved community activities that may have included rituals. Further, its location close to the Khabur River suggests that the well was not constructed to merely obtain fresh water; its major purpose could have been to guarantee the procurement of non-polluted water as this was an increasing concern among the developing Neolithic villages. On the whole, the water-well of Tell Seker al-Aheimar gives us important insights that develop our understanding of the Neolithisation processes of the region, notably the ‘domestication of water’.