Background: A recent genome-wide association study on Major Depressive Disorder (MDD) identified a specific association with a non-synonymous polymorphism (rs2522833) of a gene encoding the presynaptic protein piccolo (PCLO). A high percentage of patients who de-velop MDD have particular temperamental traits, such as passivity, pessimism, indecisiveness, and low self-esteem, which are related to the subsequent development of depression. The aims of this study were to perform a replicate case–control study and to conduct the first association study between the rs2522833 polymorphism and depression-related personality traits using the Temperament and Character Inventory (TCI) in a healthy subject sample. Methods: A total of 522 MDD patients and 375 healthy volunteers were enrolled in the study. Two hundred and forty-six controls agreed to fill out the TCI. Results: The results showed that rs2522833 CC homozygotes were more frequent among the depressed patients than in the controls (p b 0.01). The C allele distribution showed a trend in the same direction (p = 0.08). Among controls, we found that the C allele carriers were asso-ciated with personality traits increasing vulnerability to depression, including higher Harm Avoidance (HA) and lower in Novelty Seeking (NS). In particular, C allele carriers were more fearful (HA2) and fatigable (HA4), and less impulsive/more deliberate (NS2) and less extrava-gant/more frugal (NS3). Limitations: The absence of possible epistatic interaction effect. Conclusions: These results provide further support for the involvement of the PCLO gene in MDD and show that this effect may be mediated by influencing personality traits that increase the risk of major depression.