Temperature and trace metals are common environmental stressors, and their importance is increasing due to global climate change and anthropogenic pollution. The aim of the present study was to investigate whether acclimation to elevated temperature affects the response of the European bullhead (Cottus gobio) to subsequent cadmium (Cd) exposure by using enzymatic and proteomic approaches. Fish acclimated to 15 (standard temperature), 18 or 21. °C for 28 days were exposed to 1. mg Cd/L for 4 days at the respective acclimation temperature. First, exposure to Cd significantly decreased the activity of the lactate dehydrogenase (LDH) in gills of fish acclimated to 15 or 18. °C. However, an acclimation to 21. °C suppressed the inhibitory effect of Cd. Second, using a proteomic analysis by 2D-DIGE, we observed that thermal acclimation was the first parameter affecting the protein expression profile in gills of C. gobio, while subsequent Cd exposure seemed to attenuate this temperature effect. Moreover, our results showed opposite effects of these two environmental stressors at protein expression level. From the 52 protein spots displaying significant interaction effects of temperature and Cd exposure, a total of 28 different proteins were identified using nano LC-MS/MS and the Peptide and Protein Prophet algorithms of Scaffold software. The identified differentially expressed proteins can be categorized into diverse functional classes, related to protein turnover, folding and chaperoning, metabolic process, ion transport, cell signaling and cytoskeleton. Within a same functional class, we further reported that several proteins displayed reverse responses following sequential exposure to heat and Cd. This work provides insights into the molecular pathways potentially involved in heat acclimation process and the interactive effects of temperature and Cd stress in ectothermic vertebrates.