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Nitric oxide (NO) is a redox-dependent signaling molecule that plays a crucial role in regulating a wide range of biological processes in plants. It functions by post-translationally modifying proteins, primarily through S-nitrosation. Thioredoxin (Trx), a small and ubiquitous protein with multifunctional properties, plays a pivotal role in the antioxidant defense system. However, the regulatory mechanism governing the response of tomato Trxh (SlTrxh) to excessive nitrate stress remains unknown. In this study, overexpression or silencing of SlTrxhin tomato led to increased or decreased nitrate stress tolerance, respectively. The overexpression of SlTrxhresulted in a reduction in levels of reactive oxygen species (ROS) and an increase in S-nitrosothiol (SNO) contents; conversely, silencing SlTrxhexhibited the opposite trend. The level of S-nitrosated SlTrxh was increased and decreased in SlTrxhoverexpression and RNAi plants after nitrate treatment, respectively. SlTrxh was found to be susceptible to S-nitrosation both in vivoand in vitro, with Cysteine 54 potentially being the key site for S-nitrosation. Protein interaction assays revealed that SlTrxh physically interacts with SlGrx9, and this interaction is strengthened by S-nitrosation. Moreover, a combination of yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR), and transient expression assays confirmed the direct binding of SlMYB86 to the SlTrxhpromoter, thereby enhancing its expression. SlMYB86 is located in the nucleus and SlMYB86overexpressed and knockout tomato lines showed enhanced and decreased nitrate stress tolerance, respectively. Our findings indicate that SlTrxh functions downstream of SlMYB86 and highlight the potential significance of S-nitrosation of SlTrxh in modulating its function under nitrate stress.