Fibulin-3 is a member of the fibulin family that has been newly recognized as extracellular matrix proteins. We assessed the effects of fibulin-3 overexpression on chondrocyte differentiation using the clonal murine cell line ATDC5. The ATDC5-FBLN3 stably expressing fibulin-3 protein was spindle-shaped cell compared to the ATDC5-mock with plump cell. The cell growth in the ATDC5-FBLN3 was accelerated in comparison to that in the ATDC5-mock. The ATDC5-FBLN3 was not stained by Alcian blue, nor was there any cartilage aggregate formed after the induction of chondrogenic differentiation. The expression of type II collagen, aggrecan, and type X collagen was completely suppressed in ATDC5-FBLN3 even after the induction of differentiation. The overexpression of fibulin-3 reduced the expression of Sox5 and Sox6, while it maintained the expression of Sox9. These findings suggest that fibulin-3 may play an important role as a negative regulator of chondrocyte differentiation. (C) 2009 Elsevier Inc. All rights reserved.