In order to progress in the understanding of mechanical stress generation, the mesoporosity of the cell wall and its changes during maturation of poplar (Populus deltoides x P. nigra) tension wood (TW) and opposite wood (OW) were measured by nitrogen adsorption–desorption. Variations in the thickness of the gelatinous layer (G-layer) were also measured to clarify whether the mesoporosity change simultaneously with the deposition of the G-layer in TW. Results show that mesoporous structures of TW and OW were very similar in early development stages before the deposition of G-layers. With the formation of the S2 layer in OW and the G-layer in TW, the mesopore volume decreased steeply before lignification. However, in TW only, the decrease in mesopore volume occurred together with the pore shape change and a progressive increase in pore size. The different patterns observed in TW revealed that pores from G-layers appear with a different shape compared to those of the compound middle lamella, and their size increases during the maturation process until stabilising in mature wood. This observation strongly supports the hypothesis of the swelling of the G-layer matrix during maturation as the origin of maturation stress in poplar tension wood.