Soybean plants are salinity (NaCl) sensitive, with their yield significantly decreased under moderately saline conditions. GmSALT3 is the dominant gene underlying a major QTL for salt tolerance in soybean. GmSALT3 encodes a transmembrane protein belonging to the plant cation/proton exchanger (CHX) family. It is currently unknown through which molecular mechanism(s) the ER-localised GmSALT3 contributes to salinity tolerance, as its localisation excludes direct involvement in ion exclusion. In order to gain insights into potential molecular mechanism(s), we used RNA-seq analysis of roots from two soybean NILs (Near Isogenic Lines); NIL-S (salt-sensitive, Gmsalt3) and NIL-T (salt-tolerant, GmSALT3), grown under control and saline conditions (200 mM NaCl) at three time points (0h, 6h, and 3 days). Gene ontology (GO) analysis showed that NIL-T has greater responses aligned to oxidation reduction. ROS were shown less abundant and scavenging enzyme activity was higher in NIL-T, consistent with the RNA-seq data. Further analysis indicated that genes related to calcium signalling, vesicle trafficking and Casparian strip (CS) development were upregulated in NIL-T following salt treatment. We propose that GmSALT3 improves the ability of NIL-T to cope with saline stress through preventing ROS overaccumulation in roots, and potentially modulating Ca2+ signalling, vesicle trafficking and formation of diffusion barriers.