Serum levels of vitamin D levels are commonly reduced in patients with rheumatoid arthritis (RA) and have been implicated in disease pathogenesis. We recently identified a new vitamin D receptor transcriptional signature in synovial tissues from rats with mild and nonerosive arthritis, suggesting a vitamin D-mediated protective effect. In the present study, we address the hypothesis that part of the vitamin D protective effect is mediated via interference with fibroblast-like synoviocyte (FLS) invasive properties, an in vitro cellular phenotype that correlates with radiographic and histological damage in pristane-induced arthritis and RA. FLSs derived from DA rats with pristane-induced arthritis and RA patients were studied in an in vitro model of invasion through a collagen-rich barrier (Matrigel) over a 24-h period, in the presence or absence of calcitriol, an active form of vitamin D. Matrix metalloprotease (MMP) expression levels were analyzed with zymography and quantitative real-time polymerase chain reaction, and the cytoskeleton was studied with immunofluorescense microscopy. Calcitriol significantly inhibited DA and RA FLS invasion by 54% and 53%, respectively. Calcitriol also reduced interleukin (IL)-1β-induced expression of MMP-1 by 95% in DA FLSs and by 73.5% in RA FLS. Calcitriol treatment reduced actin cytoskeleton reorganization, reduced polarized formation of lamellipodia and reduced colocalization of phosphorylated focal adhesion kinase (p-FAK) with lamellipodia, all consistent with reduced cell ability to move and invade. In conclusion, we identified a new effect of calcitriol in FLS invasion. This discovery suggests that the reduced serum levels of vitamin D and its metabolites commonly seen in RA might increase risk for FLS-mediated cartilage and bone invasion and erosions. Treatment with vitamin D or its analogs has the potential to become a helpful adjuvant aimed at preventing or reducing joint destruction.