To ascertain the role of pectin disassembly in fruit softening, chelated- (CSP) and sodium carbonate-soluble (SSP) pectins from plants with a pectate lyase, FaplC, or a polygalacturonase, FaPG1, downregulated by antisense transformation were characterized at the nanostructural level. Fruits from transgenic plants were firmer than the control, although FaPG1 suppression had a greater effect on firmness. Size exclusion chromatography showed that the average molecular masses of both transgenic pectins were higher than that of the control. Atomic force microscopy analysis of pectins confirmed the higher degree of polymerization as result of pectinase silencing. The mean length values for CSP chains increased from 84 nm in the control to 95.5 and 101 nm, in antisense FaplC and antisense FaPG1 samples, respectively. Similarly, SSP polyuronides were longer in transgenic fruits (61, 67.5 and 71 nm, in the control, antisense FaplC and antisense FaPG1 samples, respectively). Transgenic pectins showed a more complex structure, with a higher percentage of branched chains than the control, especially in the case of FaPG1 silenced fruits. Supramolecular pectin aggregates, supposedly formed by homogalacturonan and rhamnogalacturonan I, were more frequently observed in antisense FaPG1 samples. The larger modifications in the nanostructure of pectins in FaPG1 silenced fruits when compared with antisense pectate lyase plants correlate with the higher impact of polygalacturonase silencing on reducing strawberry fruit softening.