Fungal pathogens secrete effector molecules into host plant cells to suppress host immunity to colonize plants. Ongoing efforts are being made to identify and characterize effector proteins in many fungal plant pathogens. Nevertheless, the precise biological and biochemical functions of many effectors, such as their trafficking from the pathogen to the host, have yet to be fully understood. In this study, we show that an effector candidate, matured PstCTE1 ofPuccinia striiformisf. sp.tritici, localizes to chloroplasts when expressed inplanta. It has no conserved transit signal region that can be detected by widely accepted prediction tools including TargetP and ChloroP, it must be carrying a unique localization signal. We have shown that N-terminal tagged red fluorescent protein has no effect on the chloroplast localization of PstCTE1, suggesting a new chloroplast translocation mechanism. We also observed the entrance of the candidate effector to the chloroplast even with the construct having the intact signal peptide on the N-terminus of the transit peptide region. Possibly due to overexpression of the protein inN. benthamina, accumulation in the ER (cytoplasm) was obvious. As previously reported, PstCTE1, similar to effector proteins, may either escape from the secretory pathway by retrograde transport, or translation may occur at alternative sites. This would result in a truncated and/or non-functional signal peptide at the N-terminus in a non-host model system (Nicotiana benthamiana), if it is not re-entering the cell from the apoplast. Our study adds PstCTE1 to the pool of few candidate effectors, experimentally shown to target the chloroplast.