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Aims The objective of this study was to identify the most suitable substrate for Cannabis sativa L. cultivation based on its effects on water relations and CBD production. Methods Biomass production, physiological parameters, mineral contents, changes in the expression levels of the PIP aquaporins and the concentration of CBD was measured in Cannabis sativa L. (var. Tiborszallasi) plants cultivated on 5 substrates with different physical-chemical characteristic under controlled conditions. Results The substrates available water (AW) was the main factor affecting growth and production. The efficiency of the water use was governed fundamentally by transpiration. S1 and S3 were those in which the plants grew optimally and allows plants to invest energy in secondary metabolites production acquiring high levels of CBD. The plants grown in S2 and S5, composed by coconut fiber and perlite, showed the lowest growth in agreement with low transpiration rates which reduce the water uptake. S5 substrate, with some AW still available, is forcing plants to invest energy in improve water and nutrients transport as observed by the high levels of nutrients in planta and high PIPs expression levels. S4 plants presented the highest inflorescences production and CBD content which can be attributed to plant stress due to the low levels of AW and high pH and electrical conductivity (EC). Conclusion The absorption of water and minerals by plants has been affected by PIP-mediated water transport, playing key roles for an optimal use of the water present in the substrates with specific isoforms involved in this responses.