OBJECTIVE In addition to absorptive disturbances, inflammatory bowel diseases (IBD) perturb normal contractility of intestinal smooth muscle. Such motility disturbances may reflect both nervous alterations and increased abundance of cytokines (e.g. IL-1β, TNF-α, and IFN-γ), which impair normal intestinal smooth muscle structure and function. In a previous study, we reported that IL-1β decreased mesenteric muscular contractility, consistent with cytokine-mediated changes in contraction present in IBD. Here, we considered the impact of pro-inflammatory cytokines on human intestinal smooth muscle cell (HISMC) contractility in vitro, which might provide a method for evaluating treatments for IBD. METHODS We used an in vitro tonic contraction assay to study how HISMC contractility was affected by cell density, serum, and cytokines (IL-1β, TNF-α, and IFN-γ). MTT (3-(4, 5-dimethyl thiazolyl-2)-2, 5-diphenyltetrazolium bromide) and wound healing analyses were also used to measure cell proliferation and migration in HISMC in response to IFN-γ. RESULTS We found that IFN-γ (but not IL-1β or TNF-α) significantly depressed HISMC tonic contractility over six days. IFN-γ also decreased HISMC proliferation, migration, and smooth muscle F-actin expression in a dose-dependent manner (studied at 4 days). CONCLUSIONS Our studies indicate that IFN-γ dose and time-dependently reduces normal HISMC contractility, motility and proliferation which may contribute to dysmotility observed in GI inflammatory disorders and that IFN-γ therapeutics might restore normal HISMC contractility impaired in IBD.