Streptococcus gordonii is a commensal Gram‐positive bacterium that acts as an opportunistic pathogen that can cause apical periodontitis, endocarditis, and pneumonia. Biofilm formation of bacteria is important for the initiation and progression of such diseases. Although lipoproteins play key roles in physiological functions, the role of lipoproteins of S. gordonii in its biofilm formation has not been clearly understood. In this study, we investigated the role of lipoproteins of S. gordonii in the bacterial biofilm formation using its lipoprotein‐deficient strain (Δlgt). The S. gordonii Δlgt exhibited increased biofilm formation on the human dentin slices or on the polystyrene surfaces compared to the wild‐type strain, while its growth rate did not differ from that of the wild‐type. In addition, the S. gordonii Δlgt strain exhibited the enhanced LuxS mRNA expression and AI‐2 production, which is known to be a positive regulator of biofilm formation, compared to the wild‐type. Concordantly, the augmented biofilm formation of S. gordonii Δlgt was attenuated by an AI‐2 inhibitor, D‐ribose. In addition, lipoproteins from purified S. gordonii inhibited the biofilm formation of S. gordonii wild‐type and Δlgt. Taken together, these results suggest that lipoprotein‐deficient S. gordonii form biofilms more effectively than the wild‐type strain, which might be related to the AI‐2 quorum‐sensing system.