Viola baoshanensis Shu, Liu et Lan is a newly identified metallophyte, and its defensive strategies against heavy metals are still unclear. In the present study, we firstly constructed a root cDNA library of the plant subjected to 300muM Cd for 48h by using suppression subtractive hybridization (SSH), and 43 unique cDNA fragments were further isolated from the library. Sequence homology analysis showed that half of the identified genes were involved in general stress defense, such as antioxidative enzymes, protein degradation and stress signal transduction. After RT-PCR and RACE analysis, a Cd-responsive gene Vb40 was identified, which could deduce a novel cysteine-rich mini-protein. Meanwhile, five cyclotide precursor genes (VbCP1-VbCP5) were also identified. The Vb40 and the VbCP1-VbCP5 were further investigated by yeast expression analysis, and they could improve copper (Cu) tolerance in hosted yeast, indicating that these species-specific genes possibly functioned in V. baoshanensis heavy metals tolerance. Our results suggested that heavy metal tolerance in V. baoshanensis relied on both general and species-specific defense.