Chloroplasts evolved from a free-living cyanobacterium acquired by the ancestor of all photosynthetic eukaryotes, including algae and plants, through a single endosymbiotic event. During endosymbiotic conversion, the majority of genes in the endosymbiont were transferred to the host nucleus and many of the proteins encoded by these genes must therefore be transported into the chloroplast after translation in the cytosol. Chloroplast-targeted proteins contain a targeting signal, named the transit peptide (TP), at the N-terminus. However, the evolution of TPs is not well understood. In this study, TPs from RbcS (rubisco small subunit) were compared between lower and higher eukaryotes. Chlamydomonas reinhardtii RbcS (CrRbcS) TP was non-functional in Arabidopsis. However, inclusion of a critical sequence motif, FP-RK, from Arabidopsis thaliana RbcS (AtRbcS) TP allowed CrRbcS TP to deliver proteins into plant chloroplasts. The position of the FP-RK motif in CrRbcS TP was critical for function. The QMMVW sequence motif in CrRbcS TP was crucial for its transport activity in plants. CrRbcS TPs containing additional plant motifs remained functional in C. reinhardtii. These results suggest that TPs evolved by acquiring additional sequence motifs to support protein targeting to chloroplasts during evolution of land plants from algae.