A novel scaffold-free self-assembled cartilage construct has been generated and used to repair particular chondral defects effectively. However, the mechanisms related to the construction of these self-assembled cartilages have not yet been fully elucidated. We hypothesize that gap junction intercellular communication (GJIC) plays a critical role in the development of self-assembled constructs upon GDF-5 induction. In this study, we investigated the effect of connexin 43 (C×43) mediated GJIC on GDF-5 modulation of chondrogenesis from two aspects, cell monolayer culture and 3-D self-assembly culture. We induced cells or self-assembled constructs with chondrogenic media (CM), growth differentiation factor 5 (GDF-5) or 1-heptanol for 3 weeks. At the end of that time, the results of quantitative fluorescence redistribution after photobleaching (FRAP) assay and immunofluorescence demonstrated that GDF-5 improved both GJIC and chondrogenic differentiation to a significant degree while 1-heptanol nearly offset the expected improvements in chondrogenesis. Biochemical assay and histology showed that GDF-5 can obviously enhance GAG, C×43 and type II collagen expressions. Conversely, we also showed that while 1-heptanol weakened GAG and type II collagen expression in self-assembled constructs, it had no effect on C×43 expression. Furthermore, real-time polymerase chain reaction showed that GDF-5 enhanced GAG and type II collagen transcription while 1-heptanol reduced them, but was affectless on C×43 transcription. This suggests that the generation of scaffold-free self-assembled cartilage from human mesenchymal stem cells upon GDF-5 induction may be mediated, at least in part, via the modulation of GJIC.