Many studies have reported high p-xylene/o-xylene permselectivities with MFI zeolite membranes; however, these results were obtained at low xylene activity (<0.01). The present work investigates the vapor permeation of xylene isomers through MFI zeolite membranes at various xylene activities up to 0.033. Using MFI zeolite membranes synthesized with and without organic templates, the effects of xylene activity and membrane microstructure on separation performance in xylene vapor permeation were systematically studied. Both p-xylene and o-xylene permeances decrease with increasing xylene activity, but the decrease is more pronounced for p-xylene resulting in a reduction in p-xylene/o-xylene selectivity. The MFI zeolite membrane fabricated without template exhibits higher p-xylene selectivity than the templated membrane due to the presence of less intercrystalline gaps in the template-free zeolite layer, and the lesser decrease in p-xylene selectivity for the template-free membrane was confirmed with increasing xylene activity. A theoretical study using the transport model for MFI zeolite membrane reveals that the xylene activity dependence of xylene permeance and selectivity could be well explained by the decrease of both xylene solubility and diffusivity in zeolitic pores with increasing xylene activity. Also, the minimization of intercrystalline gaps in the zeolite layer weakens the unfavorable xylene activity dependence in xylene vapor separation.