To understand the response of freshwater organisms to rising environmental salinity, it is essential to investigate their osmo-ionic regulatory physiology. Our laboratory experiment investigated the transfer of Palaemon argentinus (Nobili, 1901) from 2‰ (control condition) to concentrated salinity (15 and 25‰) for short- (6 h), medium- (48 h), and long-term (\textgreater504 h) acclimation periods. We measured relevant parameters in the shrimp’s haemolymph, the time course of the response of branchial V–H+–ATPase (VHA), Na+, K+–ATPase (NKA), carbonic anhydrase (CA) activity, and muscle water content. Upon prolonged acclimation to 15‰ (hyper-regulating condition), shrimp reached a new steady state of haemolymph osmolality by tightly adjusting ion concentrations to levels higher than the external medium. While NKA and CA activities recovered their pre-transfer levels, the downregulation of VHA suggests other functions rather than ion uptake after prolonged acclimation to 15‰. The activity of the three transport-related enzymes remained almost unchanged at the highest salinity (isosmotic condition), leading to increasing osmotic pressure and ion concentration after prolonged acclimation to 25‰. Although the freshwater shrimp studied here retains a certain degree of tolerance to high salinity, a common trait in palaemonid shrimps, our results highlight that 25‰ represents a significant hypertonic challenge for this species.