Plant-specific BURP family proteins have a diverse subcellular localization with different functions. However, only limited studies have investigated the functions of their different domains. In the present study, the role of the N-terminal putative signal peptide in protein subcellular localization was investigated using a tobacco cell system. The results showed that SALI3-2 was present in vacuoles, whereas AtRD22 was directed to the apoplast. The N-terminal putative signal peptides of both proteins were confirmed to be the essential and critical domains for targeting the proteins to their destinations. We also demonstrate that the expression and accumulation of mGFP in tobacco cells was increased when mGFP was fused to the putative signal peptide of SALI3-2. The findings offer the potential application of this short peptide in protein production in plants.