An extract of Bidens pilosa, an anti-diabetic Asteraceae plant, has recently been reported to modulate T cell differentiation and prevent the development of non-obese diabetes (NOD) in NOD mice. In this paper, a novel bioactive polyacetylenic glucoside, cytopiloyne (1), was identified from the Bidens pilosa extract using ex vivo T cell differentiation assays based on a bioactivity-guided fractionation and isolation procedure. Its structure was elucidated as 2beta-D-glucopyranosyloxy-1-hydroxytrideca-5,7,9,11-tetrayne by various spectroscopic methods. Functional studies showed that cytopiloyne was able to inhibit the differentiation of naïve T helper (Th0) cells into type I T helper (Th1) cells but to promote the differentiation of Th0 cells into type II T helper (Th2) cell. Accordingly, cytopiloyne also suppressed IFN-gamma expression and promoted IL-4 expression in mouse splenocytes ex vivo. These results suggest that cytopiloyne functions as a T cell modulator that may directly contribute to the ethnopharmacological effect of Bidens pilosa extract on preventing diabetes. Moreover, cytopiloyne can serve as an index compound for quality control of lot-to-lot extract preparations of Bidens pilosa.