Biofilm detachment is a physiologically regulated process that facilitates the release of cells to colonize new sites and cause infections. Streptococcus mutans is one of the major inhabitants of cariogenic dental plaque biofilm. This study tested the hypothesis that S.mutans biofilm-detached cells exhibit distinct physiological properties compared with their sessile and planktonic counterparts. Biofilm-detached cells showed a longer generation time of 2.85h compared with planktonic cells (2.06h), but had higher phosphotransferase activity for sucrose and mannose (P<0.05). Compared with planktonic cells, they showed higher chlorhexidine (CHX) resistance and fourfold more adherent (P<0.05). Increased mutacin IV production in biofilm-detached cells was noted by a larger inhibition zone against Streptococcus gordonii (31.07±1.62mm vs. 25.2±1.74mm by planktonic cells; P<0.05). The expressions of genes associated with biofilm formation (gtfC and comDE) and mutacin (nlmA) were higher compared with planktonic cells (P<0.05). In many properties, biofilm-detached cells shared similarity with sessile cells except for a higher phosphotransferase activity for sucrose, glucose, and mannose, increased resistance to CHX, and elevated expression of gtfC-, comDE-, and acidurity-related gene aptD (P<0.05). Based on data obtained, the S.mutans biofilm-detached cells are partially distinct in various physiological properties compared with their planktonic and sessile counterparts.