This paper reports the development of a technique for preparing microtextured polymer substrates for cell growth and studies the response of osteoblast cells grown on these surfaces. The surfaces were manufactured with hot embossing, where a silicon micromachined printing master was pressed into a thermoplastic polymer substrate at elevated temperature, forming a regular microgroove pattern in the polymer. The grooves were approximately 5 microm deep, 4 microm wide, and had a periodicity of 34 microm. The polymer substrate was polyimide, which can be spincast and printed in its uncured form, and is mechanically rigid and chemically nonreactive after full cure. Osteoblast cells were grown on the textured polymer substrate and their responses to grooved and smooth surfaces were observed with fluorescence microscopy. Alignment and aspect ratio were analyzed for the cell body, cell nucleus, and focal adhesions. Cell membrane body, cell nucleus, and focal adhesions all strongly aligned with the microgrooves, while only the cell body shape changed on the microgrooved surface. This novel substrate preparation technique offers the opportunity for low-cost and rapid manufacture of microtextured surfaces that can be used to control cell shape and alignment.