Aim <p>This study aimed to evaluate the histological response of mandibular mature premolars with symptomatic irreversible pulpitis (SIP) following cervical pulpotomy using three bioactive materials: Mineral Trioxide Aggregate (MTA), Biodentine, and TotalFill BC RRM.</p> Materials and methods <p>40 young, healthy patients with 60 mandibular mature premolars with deep carious lesions, diagnosed with SIP and scheduled for orthodontic extraction, were included. Premolars were randomly assigned to three groups based on the bioactive material used during pulpotomy (MTA, Biodentine, or TotalFill BC RRM). Each group was divided into two subgroups according to the follow-up period: the first subgroup was extracted after 1 week, and the second after 6 months. Histological evaluations assessed pulpal inflammation, the degree of pulp disorganization, and dentinal-like bridge formation. Kruskal-Wallis and Wilcoxon tests were used to analyse the data. The significance level was set at α = 0.05.</p> Results <p>No significant differences were observed among the bioactive materials in histological evaluations at the two time points (<i>p</i> &gt; 0.005). Most samples exhibited an inflammatory response and marked histological disruption during the first week. Over the six-month follow-up period, inflammation and histological disruption decreased progressively compared with the one-week follow-up (<i>p</i> &lt; 0.05). Moreover, a noticeable dentinal-like bridge was observed in most samples after six months, compared with the one-week follow-up (<i>p</i> &lt; 0.001).</p> Conclusion <p>The histological response following cervical pulpotomy with bioactive materials (MTA, Biodentine, and TotalFill BC RRM) in permanent mature teeth with SIP is comparable, indicating their similar effectiveness.</p> Trial registration <p>The trial protocol was registered retrospectively at the Registry of ISRCTN (Trial ID: ISRCTN16268487) on 17/03/2025.</p>