The voltage-dependent anion selective channel (VDAC) is the major porin of the mitochondrial outer membrane. Circular dichroism, infrared spectroscopy, and structure predictions indicate that VDACs form transmembrane β-barrels similar to the bacterial outer membrane proteins (OMPs). Here we have investigated the membrane insertion and folding of human VDAC isoform 1 (hVDAC1). VDAC, overexpressed in E. coli was purified from inclusion bodies in unfolded form in 8 M urea. Folding of VDAC into lipid bilayers and detergent micelles was initiated by dilution of the urea in presence of preformed lipid vesicles or detergent micelles. Insertion and folding of VDAC were monitored by fluorescence and circular dichroism spectroscopy, by protease digestion, and by single-channel recordings. After dilution of the denaturant urea, VDAC spontaneously folded into lipid bilayers of dicapryl, dilauroyl and dimyristoyl phosphatidylcholines. Refolding of VDAC into lipid bilayers was found to be near quantitative by density gradient centrifugation analysis and CD spectroscopy. Analyses of the CD spectra showed that the composition of secondary structure of refolded VDAC was comparable to that of natively isolated VDAC. Fluorescence measurements indicated that the kinetics of refolding of VDAC is composed of a fast and a slow phase. Folding conditions for VDAC, such as optimal pH, optimal refolding temperature and limiting hydrophobic thickness of the bilayer were investigated. Functional refolding of VDAC was confirmed by single channel recordings. The conductivity of refolded VDAC also showed Gaussian voltage dependence as reported previously for natively isolated VDAC.