Objective: The aim of this study was to evaluate the effect of an adhesive loaded with 0.2 % copper (Cu) and 5 % zinc oxide (ZnO) nanoparticles (Nps) on its adhesive properties and enzymatic activity at the hybrid layer ex vivo in a randomized clinical model. Methods: Fifteen patients participated in this study, and a total of 30 third molars were used. Occlusal cavities (4 x 4 x 2 mm) were made in each tooth, and randomly divided into 2 groups: (i) Experimental group: commercial adhesive loaded with 0.2wt % CuNps and 5wt % ZnONps; and (ii) Control Group: non-loaded commercial adhesive. Teeth were restored with resin composite. Thirty days later, extractions were performed. Extracted teeth were longitudinally sectioned. Nps in powder were characterized by field emission scanning electron microscope (FE-SEM) and energy dispersive X-ray (EDX) analysis. Microtensile bond strength (mu TBS), degree of conversion (DC), and nanoleakeage (NL) tests were executed. In situ zymography (Zym) was performed to evaluate the gelatinolytic activity at the hybrid layer. Student's t-test (alpha = 0.05) was applied for all tests. Results: mu TBS and DC did not show significant differences (p > 0.05) between both groups. However, NL and gelatinolytic activity at the hybrid layer showed significant values (p < 0.05) for experimental group in comparison with control group. Conclusion: The addition of 0.2 % CuNps and 5 % ZnONps to a universal adhesive decreases NL and gelatinolytic activity at the hybrid layer, without jeopardizing its adhesive properties. Significance: This randomized clinical trial with ex vivo analysis demonstrate that a commercial adhesive modified with 0.2wt % Cu and 5wt % ZnO Nps that does not affect its adhesive properties, reducing gelatinolytic activity and nanoleakage at the hybrid layer, which should contribute to an improvement of long term bondingdentine clinical performance.