Summary Ammonia production from urea by ureolytic oral bacteria is believed to have a significant impact on oral health and ecological balance of oral microbial populations. Actinomyces naeslundii is an important ureolytic organism in the oral cavity. In this study, we aimed to investigate the substrate affinity and pH optimum for ureolysis of A. naeslundii (ATCC12104), and expression of urease under different environmental factors. In addition, in vitro acid killing and pH drop experiments were used to detect the role of ureolysis in bacterial aciduricity and capacity to modulate pH homeostasis. We observed the K s value of the ureolytic activity was 7.5 mM and a pH optimum near 6.5. Urease expression by A. naeslundii (ATCC12104) was affected by multiple factors, including environmental pH, glucose and nitrogen availability. The cells could be protected against acid killing through hydrolysis of physiologically relevant concentrations of urea. A. naeslundii (ATCC12104) demonstrated a significant capacity to temper glycolytic acidification in vitro at urea concentrations normally found in the oral cavity.