PDL (periodontal ligament) is a source of multi-potent stem cells in humans and their differentiation potential to a cementoblast and osteoblast phenotypes has been shown. Tissue construction from PDL-derived cells could be considered as a valuable technique for periodontal regenerative medicine. On these basis, we determined the role of CEMP1 (cementum protein 1) as a factor to induce differentiation of human PDL cells in a 3D (three-dimensional) fashion. Human PDL cells were grown in an RCCS (rotary cell culture system) D-410 RWV (rotating wall vessel) bioreactor, and maintained in either experimental (CEMP1 2.5 μg/ml) or control media during 4 weeks. Cell proliferation in the presence of CEMP1 was determined. The tissue-like structure formed was analysed histologically, stained with Alizarin Red and Alcian Blue. ALP (alkaline phosphatase)-specific activity, immunostaining, RT—PCR (reverse transcription—PCR) and Western blotting were performed to determine the expression of BSP (bone sialoprotein), enamel [AMBN (ameloblastin) and AMEL (amelogenin)], cementum [CAP (cementum attachment protein) and CEMP1] and cartilage-related proteins (Sox9, aggrecan, types II and X collagens). Our results show that hrCEMP1 (human recombinant CEMP1) promoted cell proliferation by human PDL cells in 3D cultures and induced the formation of a tissue-like structure resembling bone and/or cementum and material similar to cartilage. The addition of hrCEMP1 to the 3D human PDL cell cultures increased ALP-specific activity by 2.0-fold and induced the expression of markers for the osteogenic, cementogenic and chondrogenic phenotypes at the mRNA and protein levels. Our data show that human PDL cells in 3D cultures with the addition of CEMP1 has the potential to be used for the bioengineering reconstruction of periodontal tissues and cartilage since our results suggest that CEMP1 stimulates human PDL cells to differentiate towards different phenotypes.