Inducible costimulator (ICOS), a prototypic T cell costimulator, is induced on activated T cells. ICOS regulates T cell activation and Th cell differentiation and is principally involved in humoral immune responses. Previous work showed that T cell accessory function is modulated by the plant-derived cannabinoid, delta-9-tetrahydrocannabinol (Δ9-THC). In light of an emerging role by ICOS in T cell-mediated immunity, the objective of this study was to investigate the effect of Δ9-THC on ICOS in activated mouse T cells. Induction of ICOS mRNA levels by phorbol ester (PMA) plus ionomycin (Io) activation in mouse splenocytes was attenuated by Δ9-THC in a concentration-related manner. Similar results were obtained in the mouse T cell line, EL4.IL-2. Anti-CD3/CD28 induced ICOS expression on CD4+ splenic T cells, which was suppressed by Δ9-THC in a time- and concentration-related manner. The PMA/Io-induced icos promoter luciferase reporter activity was also down-regulated by Δ9-THC, suggesting that the suppression of ICOS expression by Δ9-THC occurs at the transcriptional level. Moreover, transcriptional activation of the NFAT was also down-regulated by Δ9-THC as shown by a NFAT luciferase reporter assay, which is consistent with a putative role of NFAT in regulating ICOS expression. Collectively, Δ9-THC suppresses ICOS expression in activated T cells, and this suppression may be related, in part, to its modulation of NFAT signaling. The emerging role of ICOS in a wide range of immune-related diseases also suggests that it may represent a potential therapeutic target, which could be modulated by cannabinoid compounds.