Dikerogammarus villosusis an invasive Ponto-Caspian amphipod, known to have colonized most of the major European freshwater bodies in the last few decades. Several studies assessing its tolerance related to higher temperatures have been performed; however, no records are present about its response to subzero temperatures. Therefore, in this study, we wanted to experimentally assess for the first time the tolerance ofD. villosusto temperatures below 0°C. The animals were exposed to 0°C, -8°C, and room temperature for 2 and 24 hours, tracking their possible movements during the ice melting. Only long exposures at -8°C appeared to be fatal. These results provide new insights for more accurate predictive models regarding the future expansion of this invader.