Plasma membrane intrinsic proteins (PIPs), which belong to the aquaporin (AQP) family within the major intrinsic protein (MIP) superfamily, are known for the high water transport activity at the cell membrane. The subfamily is highly conservative and only includes two phylogenetic groups named PIP1 and PIP2. Natural rubber, an important industrial raw material and strategic material, is mainly derived from Para rubber tree (Hevea brasiliensis Muell. Arg.), a big tree originated in the Amazon basin of South America. As a special tissue for rubber synthesis and storage, the laticiferous cells possess no plasmodesmata with neighboring parenchyma cells, and the water balance is mainly mediated by PIP aquaporins. In previous studies, a highly abundant PIP gene denoted HbPIP1;4 was identified, however, no detectable water transport activity was found when heterologously expressed in Xenopus laevis oocytes, which may be due to its inability to localize to the cell membrane. To uncover the acting site and multimerization features of HbPIP1;4 in vivo, recombinant plasmids pNC-Cam1304-SubN-HbPIP1;4, pNC-BiFC-ECN-HbPIP1;4, and pNC-BiFC-ENN-HbPIP1;4 for subcellular localization and bimolecular fluorescence complementation (BiFC) were successfully constructed. Agrobacterium tumefaciens transformed with above recombinant plasmids were used to infiltrate tobacco (Nicotiana benthamiana), and transiently transformed leaves were subsequently checked using laser confocal microscopy. Compared with wide distribution of fluorescence signals throughout the cell for the control pNC-Cam1304-SubN, signals of pNC-Cam1304-GFP-HbPIP1;4 were restricted to the cell membrane, supporting the plasma membrane localization of HbPIP1;4, which is consistent with the bioinformatics prediction. The result was further supported by the BiFC analysis, which also implied that HbPIP1;4 could form homomultimer, in accordance with the 3D prediction via homology modeling. The results would lay a solid foundation for further uncovering the mechanism of water balance of the laticifer. Nevertheless, PIP aquaporins that could interact with HbPIP1;4 in rubber tree remain to be further characterized.