Background Plant adaptation to environmental stress is crucial for improving crop resilience and productivity. The growth and yield of Isatis indigotica are significantly affected by water conditions. In this study, high-throughput transcriptome sequencing was performed on leaf samples from Isatis indigotica after different treatments: normal precipitation (CK), 40% rainfall reduction (R1), 80% rainfall reduction (R2), 40% rainfall enhancement (I1) and 80% rainfall enhancement (I2). Results Under 80% rainfall augmentation (I2), the malondialdehyde (MDA) content of Isatis indigotica leaves was the lowest, and the proline (pro) and catalase (CAT) activities were the highest. These findings indicate that normal precipitation conditions do not meet the optimal water requirements for the growth of Isatis indigotica and that appropriate irrigation can be used to improve the accumulation and quality of medicinal substances from this species. Transcriptome analysis of Isatis indigotica leaves compared with those in the control group (CK) revealed 896, 2551, 1294, and 3082 differentially expressed genes in the reduced rainfall reduction groups (R1, R2) and increased rainfall groups (I1, 12), respectively. The number of differentially expressed genes (DEGs) gradually increased with increasing rainfall and decreased after rainfall reduction. The GO enrichment results revealed that the DEGs were significantly enriched in functions such as cellular processes, metabolic processes, stimulus response, cell structure, and catalytic and binding activities. KEGG analysis revealed that metabolic pathways such as glutathione metabolism, phenylpropanoid biosynthesis, and plant hormone signaling were significantly enriched, with the greatest number of enriched genes. This study revealed 32 antioxidant system-related genes, 49 phenylpropanoid biosynthesis-related genes, and 49 plant hormone signaling pathway-related genes among the significantly enriched pathways. Conclusions This study provides new insights into the regulation of Isatis indigotica leaves in response to different water contents at the molecular level. The findings also provide a reference for optimizing the field management of Isatis indigotica and improving the quality and yield of medicinal materials.