Objectives <p>This in vitro study aimed to evaluate the effect of different application protocols of surface pre-reacted glass-ionomer (S-PRG) sealant on penetration ability, retention, and occlusal caries inhibition, in comparison with resin-based sealant (RBS) under simulated oral conditions.</p> Materials and methods <p>Eighty-four extracted human third molars were randomly assigned to five groups: S-PRG sealant applied per manufacturer’s instructions (G1), S-PRG with 37% phosphoric acid etching pretreatment (G2), S-PRG with fluoride varnish pretreatment (G3), RBS (G4), and a no-sealant control (G5). Sealant retention was assessed through visual inspection following water storage at 37&#xa0;°C and thermocycling, whereas penetration depth was evaluated using scanning electron microscopy (SEM). Caries inhibition was evaluated via mineral density changes (ΔMDV) measured by micro-CT after 21-day pH cycling and <i>Streptococcus mutans</i> bacterial challenge. Statistical analysis included Kruskal-Wallis tests, Mann-Whitney U tests with Bonferroni correction, Chi-square tests, Spearman’s correlation, and multiple linear regression analyses.</p> Results <p>No statistically significant differences in sealant retention were found among the groups. RBS (G4) exhibited the highest median penetration (100%), followed by G2 (85.0%), G1 (59.5%), and G3 (44.5%), with significant differences among specific pairwise comparisons (<i>p</i> &lt; 0.001). No significant difference in ΔMDV were observed in pH cycling, but bacterial testing revealed significantly lower ΔMDV in G2 and G4 compared to G3 and G5 (<i>p</i> ≤ 0.005).</p> Conclusions <p>S-PRG sealants pretreated with phosphoric acid etching demonstrated significantly greater caries inhibition effects compared to using self-etched primer alone, and its caries prevention efficacies were comparable to that of RBS.</p> Clinical relevance <p>These in vitro findings support the use of phosphoric acid etching to enhance the performance of S-PRG sealants.</p>