Rhizobia are soil proteobacteria able to establish nitrogen-fixing symbiosis with host legumes. This symbiotic interaction, which is highly important from ecological and agronomical points of view since it allows growth of legumes in soils poor in nitrogen, requires a complex interchange of molecular signals between both symbionts. The production of rhizobial molecular signals is elicited by flavonoids, which are phenolic compounds exuded by legume roots. Recent work has shown that osmotic stress can also promote the formation of symbiotic signals in some rhizobia. In this work, we show that this is also the case for Sinorhizobium fredii HH103, a rhizobial strain able to establish symbiosis with hundreds of legumes, including the very important crop, soybean. Non-ionic osmotic stress, which can be encountered by the bacterium in the rhizosphere or inside the legume host, affected the expression of hundreds of bacterial genes and, consequently, influenced diverse bacterial traits, including the production of symbiotic signals and certain characteristics that may be relevant for successful interaction with the host: motility, production of the phytohormone indole acetic acid, and production of molecules involved in bacterial cell-to-cell communication. Thus, our work provides new evidence of how stress can promote rhizobia-legume symbiosis. (1) Background: Some rhizobia, such as Rhizobium tropici CIAT 899, activate nodulation genes when grown under osmotic stress. This work aims to determine whether this phenomenon also takes place in Sinorhizobium fredii HH103. (2) Methods: HH103 was grown with and without 400 mM mannitol. β-galactosidase assays, nodulation factor extraction, purification and identification by mass spectrometry, transcriptomics by RNA sequencing, motility assays, analysis of acyl-homoserine lactones, and indole acetic acid quantification were performed. (3) Results: Non-ionic osmotic stress induced the production of nodulation factors. Forty-two different factors were detected, compared to 14 found in the absence of mannitol. Transcriptomics indicated that hundreds of genes were either activated or repressed upon non-ionic osmotic stress. The presence of 400 mM mannitol induced the production of indole acetic acid and acyl homoserine lactones, abolished swimming, and promoted surface motility. (4) Conclusions: In this work, we show that non-ionic stress in S. fredii HH103, caused by growth in the presence of 400 mM mannitol, provokes notable changes not only in gene expression but also in various bacterial traits, including the production of nodulation factors and other symbiotic signals.