The macroalga Ulva limnetica K. Ichihara et S. Shimada is the only known Ulva species to be distributed exclusively in freshwater and is restricted to freshwater bodies in the Ryuku archipelago. Molecular phylogenetic analysis suggests that U. limnetica originally evolved from marine forms of Ulva. The mechanisms of adaptation to freshwater in Ulva spp. are poorly understood. In this study, we isolated genes potentially involved in adaptation or tolerance to freshwater conditions in U. limnetica, using suppression subtractive hybridization between mRNAs of samples cultured in freshwater and seawater conditions. A total of 219 genes, up-regulated by the exposure of the macroalga to freshwater, were isolated. Reverse transcription–PCR (RT–PCR) revealed 39 clones, including malate dehydrogenase, soluble starch synthase, triosephosphate isomerase, plastid ribosomal protein, DnaJ-like protein, and chloroplast ascorbate peroxidase (APX), which were specifically or preferentially expressed in freshwater conditions. These 39 clones were also analyzed for their temporal transcriptional response to freshwater conditions. A large majority of these up-regulated genes showed a transient peak of expression after 1–4 h, followed in the next 24 h by a decrease to a stable level (over the 7 d of the experiment). After the initial response peak, the level of expression either remained higher than in the control (long-term response) or returned to a level similar to pretreatment level. A few genes showed a more delayed response (i.e., after several days) to freshwater exposure. Finally, we discussed the possible contributions of the freshwater-induced genes in the acquisition of freshwater adaptation or tolerance of U. limnetica.