Gold nanoparticles (AuNPs) with surface-anchored molecules present tremendous potential in tissue regeneration. However, little is known about chiral-modified AuNPs. In this study, we successfully prepared L/D-cysteine-anchored AuNPs (L/D-Cys-AuNPs) and studied the effects of chiral-modified AuNPs on osteogenic differentiation and autophagy of human periodontal ligament cells (hPDLCs) and periodontal tissue regeneration. In vitro, more L-Cys-AuNPs than D-Cys-AuNPs tend to internalize in hPDLCs. L-Cys-AuNPs also significantly increased the expression of alkaline phosphatase, collagen type 1, osteocalcin, runt-related transcription factor 2, and microtubule-associated protein light chain 3 II and decreased the expression of sequestosome 1 in hPDLCs compared to the expression levels in the hPDLCs treated by D-Cys-AuNPs. In vivo tests in a rat periodontal-defect model showed that L-Cys-AuNPs had the greatest effect on periodontal-tissue regeneration. The activation of autophagy in L-Cys-AuNP-treated hPDLCs may be responsible for the cell differentiation and tissue regeneration. Therefore, compared to D-Cys-AuNPs, L-Cys-AuNPs show a better performance in cellular internalization, regulation of autophagy, cell osteogenic differentiation, and periodontal tissue regeneration. This demonstrates the immense potential of L-Cys-AuNPs for periodontal regeneration and provides a new insight into chirally modified bioactive nanomaterials.