A chitosan-collagen sponge is biocompatible and osteoconductive. It is hypothesized that collagen in chitosan-collagen sponge will promote growth and differentiation of osteoblasts. Objectives: This study aimed to investigate attachment, growth and differentiation of osteoblasts on chitosan-collagen sponges (3D) ratio 1:1 and 1:2. Chitosan, collagen sponges, and cell culture plate (2D) were tested as controls. Methods: MC3T3-E1 cells, 1x105 cells, were seeded on each 5x3mm sponge and cultivated in mineralized culture medium for 27 days. Metabolic activity of cells (MTT assay), alkaline phosphatase activity (ALP) were detected at 24 hours after seeding and every 3 day. Calcium content assay and scanning electron microscope (SEM) were detected at 15, 21, 27 days after cell seeding. Differences among the experiment groups were tested by ANOVA and multiple comparison was done by Scheffe or Dunnette's T3 method if it was applicable, p value was set at 0.05. Results: It is revealed that chitosan-collagen 1:1 and 1:2 sponges had porous structure size 100–150 µm. SEM showed well attachment and growth of cells on sponges of all groups. MTT assay showed that growth of cells on chitosan sponges was significantly higher (p<0.05) than others at 12-18 days and there was no significant difference among other groups. During 18–27 days, collagen demonstrated the highest ALP level, followed by chitosan-collagen 1:2 and 1:1 groups. Calcium level was markedly highest in collagen group at 15-27 days (p<0.05) but was lower level in other groups and not significant difference. Cells on 2D plates posed the highest level of growth and lowest ALP levels compared to cells on scaffolds of all groups. Conclusions: The result indicates that collagen improved osteoconductive property of chitosan scaffolds in supporting osteoblastic differentiation. Chitosan-collagen scaffolds support growth and differentiation of osteoblasts. This study was supported by graduate school, Faculty of Dentistry, Prince of Songkhla University.