Aim: To assess the in vitro effect of keratin preparations on the viability and proliferation of cementoblast OCCM-30 and fibroblast L929 cells and on the mineralization capability of the OCCM-30 cells. Methodology: Cells of L929 and OCCM-30 were assessed for viability in growth media supplemented with 10, 1 and 0.1 mg/ml of keratin using a LIVE/ DEAD assay and confocal laser scanning microscopy. Cell proliferation was tested with similar keratin concentrations using an alamarBlue® proliferation assay at 0, 24, 48 and 72 h. Alkaline phosphatase assay was performed for OCCM-30 cells with keratin concentrations of 1 mg/ml, 0.1 mg/ml at 3, 6, and 10 days and an alizarin red assay was performed at 14 days. Results: The viability assay showed concentrations at 1 mg/ml or greater of keratin proved toxic to L929 cells. The proliferation assay for OCCM-30 cells showed keratin concentration at 10 mg/ml prevented its proliferation and was significantly lower than the other keratin groups (P < 0.0001). There was significantly higher OCCM-30 cell ALP activity with the control and 0.1 mg/ml groups compared with the 1 mg/ml group at 6 d and 12 days (P < 0.0001). The alizarin red assay was consistent with the ALP activity of the OCCM-30 cells. Conclusion: Low concentration keratin preparations were shown to allow normal cell proliferation of L929 and OCCM-30 cells and normal mineralization of the cementoblasts.