Purpose: To investigate whether interfacial enzymatic activity and adhesion receptiveness of artificial caries-affected dentin (ACAD) simulate those of natural caries-affected dentin (NCAD). Materials and Methods: Thirty dentin specimens were prepared from human molars to determine interfacial gelatinolytic activity using in situ zymography and adhesion experiments (micropermeability and bond strength [µTBS]). Groups were formed according to the type of dentin: artificial caries-affected dentin (ACAD), natural caries-affected dentin (NCAD), or sound dentin. ACAD was produced by incubating dentin with Streptococcus mutans in a chemically defined medium (CDM) with 1% sucrose for 7 days at 37°C under anaerobic conditions. CDM was replaced daily, and the sterility as well as the pH of the culture was monitored. Adhesion experiments employed Single Bond Universal (3M Oral Care) in self-etch mode. Data were individually processed and analyzed using ANOVA and post-hoc tests (α = 0.05). Results: The enzymatic activity of ACAD was similar to that of sound dentin, but was lower than that of NCAD, which elicited the highest activity (p 0.05). Interfacial micropermeability intensity at the hybrid layer or in underlying dentin (5 µm below the interface) was similar in all types of dentin (p > 0.05). On the other hand, substrate permeability was higher for NCAD than for ACAD. The highest sealing ability was detected in sound dentin. Bond strengths to ACAD were higher than to NCAD. However, the highest µTBS was observed in sound dentin (p 0.05). Conclusion: Artificial caries-affected dentin simulated neither the gelatinolytic activity nor bonding receptiveness of natural caries-affected dentin. Keywords: enzymatic activity, micropermeability, caries-affected dentin, resin adhesion