SummaryThe application of mineral trioxide aggregates (MTA) cement during the root canal obturation is gaining concern due to its bioactive characteristic to form an apatite in dentinal tubules. In this regard, this study was to assess the biomineralization of dentinal tubules following root canal obturation by using pozzolan‐based (Pz‐) MTA sealer cement (EndoSeal MTA, Maruchi). Sixty curved roots (mesiobuccal, distobuccal) from human maxillary molars were instrumented and prepared for root canal obturation. The canals were obturated with gutta‐percha (GP) and Pz‐MTA sealer by using continuous wave of condensation technique. Canals obturated solely with ProRoot MTA (Dentsply Tulsa Dental) or Pz‐MTA sealer were used for comparison. In order to evaluate the biomineralization ability under different conditions, the PBS pretreatment before the root canal obturation was performed in each additional samples. At dentin‐material interfaces, the extension of intratubular biomineralization was analyzed using scanning electron microscopy (SEM) and energy dispersive spectroscopy. When the root canal was obturated with GP and Pz‐MTA sealer, enhanced biomineralization of the dentinal tubules beyond the penetrated sealer tag was confirmed under the SEM observation (p < 0.05). Mineralized apatite structures (calcium/phosphorous ratio, 1.45–1.89) connecting its way through the dentinal tubules were detected at 350–400 μm from the tubule orifice, and the pre‐crystallization seeds were also observed along the intra‐ and/or inter‐tubular collagen fiber. Intratubular biomineralization depth was significantly enhanced in all PBS pretreated canals (p < 0.05). Pz‐MTA cement can be used as a promising bioactive root canal sealer to enhance biomineralization of dentinal tubules under controlled environment. SCANNING 38:50–56, 2016. © 2015 The Authors. Scanning Published by Wiley Periodicals, Inc.