Aquaporins facilitate water flux across biomembranes in cells and are involved in various physiological phenomena in several plant tissues. Generally, the water-flux activity of exogenously expressed aquaporins is measured in Xenopus laevis oocytes or yeast. However, heterogeneous systems are not likely to be optimal for plant aquaporin analysis. Thus, we created a new experimental system for functional analysis of plant aquaporins using lily (Lilium longiflorum) pollen protoplasts. Large protoplasts with uniform diameters of approximately 95 mm were isolated from lily pollen grains. No plasma membrane intrinsic protein (PIP) aquaporin was detected in lily pollen. For ectopic expression of PIPs in the lily pollen protoplasts, we constructed plasmids in which Arabidopsis AtPIP1;1 or AtPIP2;1 was under the control of a strong pollen-specific promoter (maize Zm13). The PCR-amplified DNA fragments were transformed into the pollen protoplasts by electroporation. Between 45 and 60% of protoplasts were successfully transformed. The protoplasts expressing AtPIP2;1 significantly increased in volume in a hypotonic solution (350 mM mannitol), compared with the vector control. In contrast, the changes in volume of the protoplasts expressing AtPIP1;1 were similar to that of the vector control. This result suggests that PIP2 induces higher water-flux activity in plant cells, whereas PIP1 does not. Thus, we propose the lily pollen protoplast as a simple and useful experimental system to analyze the function of plant aquaporins.