This study presents the effects of a novel plied yarn structure consisting of different yarn components and yarn twist levels on the Poisson's ratio and auxetic behavior of yarns. The plied yarn structures are formed with bulky and soft yarn components (helical plied yarn [HPY], braided yarn, and monofilament latex yarn) and stiff yarn components (such as high tenacity [HT] and polyvinyl chloride [PVC]-coated polyester yarns) to achieve auxetic behavior. Experimental results showed that as the level of yarn twist increased, the Poisson's ratios and the tensile modulus values of the plied yarns decreased, but the elongation values increased. A negative Poisson's ratio (NPR) was obtained in HT-latex and PVC-latex plied yarns with a low twist level. The plied yarns formed with braid-HPY and braid-braid components gave partial NPR under tension. A similar result was achieved for yarns with HT-latex and PVC-latex components. Since partial NPR was seen in novel plied yarns with braided and HPY components, it is concluded that yarns formed with bulky-bulky yarn components could give an auxetic performance under tension.