Salinity stress severely hampers plant growth and productivity. How to improve plants’ salt tolerance is an urgent issue. However, the molecular basis of plant resistance to salinity still remains unclear. In this study, we used two poplar species with different salt sensitivities to conduct RNA-sequencing and physiological and pharmacological analyses; the aim is to study the transcriptional profiles and ionic transport characteristics in the roots of the two Populus subjected to salt stress under hydroponic culture conditions. Our results show that numerous genes related to energy metabolism were highly expressed in Populus alba relative to Populus russkii, which activates vigorous metabolic processes and energy reserves for initiating a set of defense responses when suffering from salinity stress. Moreover, we found the capacity of Na+ transportation by the P. alba high-affinity K+ transporter1;2 (HKT1;2) was superior to that of P. russkii under salt stress, which enables P. alba to efficiently recycle xylem-loaded Na+ and to maintain shoot K+/Na+ homeostasis. Furthermore, the genes involved in the synthesis of ethylene and abscisic acid were up-regulated in P. alba but downregulated in P. russkii under salt stress. In P. alba, the gibberellin inactivation and auxin signaling genes with steady high transcriptions, several antioxidant enzymes activities (such as peroxidase [POD], ascorbate peroxidase [APX], and glutathione reductase [GR]), and glycine-betaine content were significantly increased under salt stress. These factors altogether confer P. alba a higher resistance to salinity, achieving a more efficient coordination between growth modulation and defense response. Our research provides significant evidence to improve the salt tolerance of crops or woody plants.