BackgroundNitrogen is very important for crop yield and quality. Crop producers face the challenge of reducing the use of mineral nitrogen while maintaining food security and other ecosystem services. The first step towards understanding the metabolic responses that could be used to improve nitrogen use efficiency is to identify the genes that are up- or downregulated under treatment with different forms and rates of nitrogen. We conducted a transcriptome analysis of barley (Hordeum vulgareL.) cv. Anni grown in a field experiment in 2019. The objective was to compare the effects of organic (cattle manure) and mineral nitrogen (NH4NO3; 0, 40, 80 kg N ha−1) fertilizers on gene activity at anthesis (BBCH60) and to associate the genes that were differentially expressed between treatment groups with metabolic pathways and biological functions.ResultsThe highest number of differentially expressed genes (8071) was found for the treatment with the highest mineral nitrogen rate. This number was 2.6 times higher than that for the group treated with a low nitrogen rate. The lowest number (500) was for the manure treatment group. Upregulated pathways in the mineral fertilizer treatment groups included biosynthesis of amino acids and ribosomal pathways. Downregulated pathways included starch and sucrose metabolism when mineral nitrogen was supplied at lower rates and carotenoid biosynthesis and phosphatidylinositol signaling at higher mineral nitrogen rates. The organic treatment group had the highest number of downregulated genes, with phenylpropanoid biosynthesis being the most significantly enriched pathway for these genes. Genes involved in starch and sucrose metabolism and plant-pathogen interaction pathways were enriched in the organic treatment group compared with the control treatment group receiving no nitrogen input.ConclusionThese findings indicate stronger responses of genes to mineral fertilizers, probably because the slow and gradual decomposition of organic fertilizers means that less nitrogen is provided. These data contribute to our understanding of the genetic regulation of barley growth under field conditions. Identification of pathways affected by different nitrogen rates and forms under field conditions could help in the development of more sustainable cropping practices and guide breeders to create varieties with low nitrogen input requirements.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-023-04263-2.