<p>L-proline is a non-essential amino acid that can be metabolised by the oral microbiome. This study hypothesised that L-proline could influence the microbiome and that shifts in the microbiome could subsequently affect non-volatile flavour perception. A semi-trained panel (n = 20) completed sensory assessments using the generalised labelled visual analogue scale (glVAS) for nine tastants at baseline, during a five-day supplementation phase, and post-supplementation within a three-week protocol. Analysis at the tastant level identified a significant increase in menthol taste perception (p = 0.011, Wilcoxon) during supplementation at the group level (n = 20). Shotgun sequencing results demonstrated a distinct beta diversity separation in metabolic pathways during supplementation at the entire group level (p = 0.008). Piano analysis suggested that increased protein synthesis could influence non-volatile flavour perception, while lipopolysaccharide (LPS) might prevent changes in non-volatile flavour perception via immune response. A mixed-effects linear regression model revealed that metabolic pathways were stronger predictors of non-volatile flavour perception than the microbiome itself. The metabolic pathways were positively associated with menthol taste and sodium chloride perception. In conclusion, L-proline supplementation affected metabolic pathways, which altered non-volatile flavour perception.</p>