This paper describes a novel cell fusion method which induces cell fusion between adhered cells through a microslit for preventing nuclear mixing. For this purpose, a microfluidic device which had approximate to 100 cell pairing structures (CPSs) making cell pairs through microslits with 2.1 +/- 0.3 mu m width was fabricated. After trapping NIH3T3 cells with hydrodynamic forces at the CPSs, the cells were fused through the microslit by the Sendai virus envelope method. With following timelapse observation, we discovered that the spread cells were much less susceptible to nuclear migration passing through the microslit compared with round cells, and that cytoplasmic fraction containing mitochondria was transferred through the microslit without nuclear mixing. These findings will provide an effective method for cell fusion without nuclear mixing, and will lead to an efficient method for reprograming and transdifferentiation of target cells toward regenerative medicine. Biotechnol. Bioeng. 2014;111: 1464-1468. (c) 2014 Wiley Periodicals, Inc.