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MicroRNAs (miRNAs) have important functions in the plant salt stress response. However, the functions of nonconserved miRNAs in soybean salt stress adaptation remain unclear. In this study, the soybean nonconserved miRNA gma-miR4359b and its target mRNA F-box gene GmFBX193 were functionally identified in response to salt stress. gma-miR4359b responded to salt stress in soybean hairy roots, stems, and leaves. Under salt stress, the root length ratio, root surface area ratio, root volume ratio, root tip number, survival rate, chlorophyll content, soluble sugar content, proline content, superoxide dismutase activity, and peroxidase enzyme activity of soybean hairy roots overexpressing gma-miR4359b (MIR4359b) were increased compared to those of the K599 (control) soybean hairy roots, while the relative electrolyte leakage and malondialdehyde content of the MIR4359b soybean hairy roots decreased. In contrast, soybean hairy roots in which target mimicry (MIM4359b) and short tandem target mimic (STTM4359b) inhibited gma-miR4359b exhibited a salt-sensitive phenotype. The transcription level of GmFBX193 was decreased in MIR4359b soybean hairy roots, while the transcription level of GmFBX193 was increased in MIM4359b and STTM4359b soybean hairy roots. GmFBX193 responded to salt stress in soybean roots, and GmFBX193 was identified as a nuclear-localized protein. Under salt stress, soybean hairy roots overexpressing GmFBX193 had a similar phenotype to MIM4359b and STTM4359b soybean hairy roots. Compared with K599 (control) soybean hairy roots, MIR4359b soybean hairy roots had 172 differentially expressed genes (DEGs), and MIM4359b soybean hairy roots had 2129 DEGs. Some known stress-related transcription factors were included among the DEGs. Taken together, these results suggest that gma-miR4359b plays a positive role in the soybean salt stress response by negatively regulating GmFBX193. • The nonconserved soybean miRNA gma-miR4359b and its target mRNA F-box gene GmFBX193 responded to salt stress. gma-miR4359b positively regulates the soybean response to salt stress by targeting GmFBX193. • gma-miR4359b is a positive regulator of salt tolerance in soybean. • GmFBX193 negatively regulates soybean tolerance to salt stress. • Transcriptome sequencing showed that gma-miR4359b could affect the transcription levels of some transcription factors related to the salt stress response. [ABSTRACT FROM AUTHOR]