One of the biggest challenges today is to reduce the application of chemical fertilizers by promoting waste recycling and obtaining organic raw materials for agriculture according to the principles of circular economy. This is due to the rapid growth of the population and food demand, which have led to higher reliance on chemical fertilizers in agriculture, leading to intensive strain on natural resources. Hence, we explored the impact of human urine derivatives (UDs) compared to commercial fertilizers on lettuce (Lactuca sativa L. cv. Grand Rapids) metabolic profile and antioxidant activity in a soilless cultivation (Nicastro et al., 2024). Furthermore, to gain a better understanding of the effects of UDs on plant metabolism by gene regulation, this research investigated their impact on gene expression, particularly on genes encoding enzymes involved in essential biochemical pathways, thus looking into possible stress responses or various regulation effects. Treatment with K-Struvite resulted in the highest expression levels of genes encoding key enzymes involved in nitrogen metabolism, nitrate reductase (NR), glutamate synthase (GOGAT X1), and glutamine synthetase (GS), with the lowest expression levels in plants treated with hydrolyzed urine. The latter, however, showed an increased expression of genes encoding stress-related enzymes like GOGAT X2, catalase (CAT), and glutamate decarboxylase (GAD). Additionally, the expression of P5CS (Δ1-pyrroline-5-carboxylate synthase), a key gene in proline synthesis, decreased across all treatments compared to the control, with the most significant reduction detected in ED concentrate and K-Struvite. Genes encoding CHS2 (chalcone synthase), AGPase (ADP glucose pyrophosphorylase), and cytGR (glutathione reductase cytosolic) enzymes showed higher expressions in plants treated with K-Struvite. Meanwhile, genes for PAL (phenylalanine ammonium lyase) and chlGR (Glutathione reductase, chloroplastic) enzymes were significantly overexpressed after treatment with ED concentrate compared to the NPK control. Fertigation with UDs in soilless cultivation of lettuce affects gene expression levels by supplying various forms of nitrogen, while also inducing various morphological and metabolic responses as a result of their distinct composition.