Stimuli-responsive polymer microspheres have attracted great attention due to their applications in drug delivery system which can offer considerable versatility for encapsulation and triggered release. In this article, thermo-sensitive poly (N-isopropyl acrylamide-co-acrylic acid) [P(NIPAM-co-AA)] microspheres with narrow size distribution were successfully prepared using a new technique-premix membrane emulsification technique combined with low temperature polymerization. Coarse W/O (water in oil) emulsion with broad size distribution was repeatedly pressed through Shirasu Porous Glass (SPG) membrane under nitrogen gas pressure to form the fine emulsion with narrow size distribution. In order to avoid phase transition triggered aggregation of thermo-sensitive P(NIPAM-co-AA) microspheres during polymerization, N, N, N' N'-tetramethylethylene-diamine (TEMED) was introduced to polymerization system by adding it into W/O emulsion to initiate polymerization at low temperature. The morphology and size distribution were investigated by confocal laser scanning microscopy (CLSM) and zeta potential analyzer. Rhodamine 123 was screened out from several dyes to label P(NIPAM-co-AA) microspheres in order to characterize them by CLSM. Effects of trans-membrane pressure, number of cross membrane passes and stirring rate during polymerization on size distribution of the microspheres were investigated in detail. The optimized preparation conditions were determined as follows: trans-membrane pressure was 250 kPa and trans-membrane passes were three times when using a membrane of 5.2 mu m pore size. The obtained microspheres were around 5.11 with the polydispersity index of 0.0312. Good linear relationship (Y = 0.89X + 0.62, R-2 = 0.999) between the membrane pore size (X) and the mean diameter of the microspheres (Y) was observed. This research would also shed light on preparation of other thermo-sensitive microspheres with narrow size distribution, which would have extensive applications in drug delivery system (DDS). (C) 2012 Elsevier B.V. All rights reserved.