Bacterial surface motility is a complex microbial trait that contributes to host colonization. However, the knowledge about regulatory mechanisms that control surface translocation in rhizobia and their role in the establishment of symbiosis with legumes is still limited. Recently, 2-tridecanone (2-TDC) was identified as an infochemical in bacteria that hampers microbial colonization of plants. In the alfalfa symbiont Sinorhizobium meliloti, 2-TDC promotes a mode of surface motility that is mostly independent of flagella. To understand the mechanism of action of 2-TDC in S. meliloti and unveil genes putatively involved in plant colonization, Tn5 transposants derived from a flagellaless strain that were impaired in 2-TDC-induced surface spreading were isolated and genetically characterized. In one of the mutants, the gene coding for the chaperone DnaJ was inactivated. Characterization of this transposant and newly obtained flagella-minus and flagella-plus dnaJ deletion mutants revealed that DnaJ is essential for surface translocation, while it plays a minor role in swimming motility. DnaJ loss-of-function reduces salt and oxidative stress tolerance in S. meliloti and hinders the establishment of efficient symbiosis by affecting nodule formation efficiency, cellular infection, and nitrogen fixation. Intriguingly, the lack of DnaJ causes more severe defects in a flagellaless background. This work highlights the role of DnaJ in the free-living and symbiotic lifestyles of S. meliloti.