Visfatin is a secretory protein which exerts insulin mimetic and proinflammatory effects, also functioning as an intracellular enzyme to produce NAD. Plasma visfatin levels and visfatin mRNA expression in adipose tissues are increased in obese subjects. Visfatin does not have a decent cleavable signal sequence, and the mechanism, that mediates release of visfatin from adipocytes, remains poorly understood. In this study, we demonstrate that visfatin is released abundantly into culture medium from 3T3-L1 adipocytes. Subcellular fractionation analysis showed that visfatin was localized in the cytosol, but not in nucleus, membrane, vesicles, or mitochondria fractions. Visfatin release was not reduced by Brefeldin A and Monensin, inhibitors of endoplasmic reticulum (ER)-Golgi-dependent secretion. In addition, visfatin was not released on microvesicles. These results suggest that visfatin should be released from 3T3-L1 adipocytes via an ER-Golgi or microvesicles independent pathway.