Curbing greenhouse gas (GHG) emissions to combat climate change is a major global challenge. Although irrigated agriculture consumes considerable energy that generates GHG emissions, the biomass produced also represents an important CO2 sink, which can counterbalance the emissions. The source of the water supply considerably influences the irrigation energy consumption and, consequently, the resulting carbon footprint. This study evaluates the potential impact on the carbon footprint of partially and fully replacing the conventional supply from Tagus–Segura water transfer (TSWT) with desalinated seawater (DSW) in the irrigation districts of the Segura River basin (south-eastern Spain). The results provide evidence that the crop GHG emissions depend largely on the water source and, consequently, its carbon footprint. In this sense, in the hypothetical scenario of the TSWT being completely replaced with DSW, GHG emissions may increase by up to 50% and the carbon balance could be reduced by 41%. However, even in this unfavourable situation, irrigated agriculture in the study area could still act as a CO2 sink with a negative total and specific carbon balance of −707,276 t CO2/year and −8.10 t CO2/ha-year, respectively. This study provides significant policy implications for understanding the water–energy–food nexus in water-scarce regions.