The proteasome is a large multiprotein complex that processes intracellular proteins functioning as cell cycle regulators and transcription factors. It has been shown that the chymotryptic component of the proteasome is an important regulator of osteoblast differentiation and bone formation, with inhibitors of the proteasome increasing osteoblast differentiation and bone formation. Yet, little is known about the effects of the proteasomal activity in the growth plate. In the present study, we cultured rat metatarsal bones in the presence of proteasome inhibitor I (PSI), a known inhibitor of the chymotrypsin-like activity of the 20S proteasome. PSI suppressed growth plate chondrocyte proliferation and hypertrophy/differentiation, and induced chondrocyte apoptosis. All these cellular effects led to reduced metatarsal linear growth. In cultured chondrocytes, PSI increased the expression of β-catenin (a negative regulator of chondrogenesis) and reduced the DNA binding of nuclear factor κB, a transcription factor that stimulates growth plate chondrogenesis. In conclusion, our findings suggest that the proteasomal activity facilitates growth plate chondrogenesis and, in turn, longitudinal bone growth.