Nanoperforated poly(lactic acid) (PLA) free-standing nanomembranes (FsNMs) have been prepared using a two-step process: (1) spin-coating a mixture of immiscible polymers to provoke phase segregation and formation of appropriated nanofeatures (i.e. phase separation domains with dimensions similar to the entire film thickness); and (2) selective solvent etching to transform such nanofeatures into nanoperforations. For this purpose, PLA has been mixed with polyethylene glycol (PEG) and poly(vinyl alcohol) (PVA). Unfortunately, the characteristics of PLA:PEG mixtures were not appropriated to prepare nanoperforated FsNMs. In contrast, perforated PLA FsNMs with pores crossing the entire film thickness, which have been characterized by scanning electron microscopy and atomic force microscopy, were obtained using PLA:PVA mixtures. The diameter (Pdbl) of such pores has been controlled through both the PLA:PVA ratio and the processing conditions of the mixtures, FsNMs with pores of Pdbl ≈ 0.8 μm, 170 nm and 65 nm being achieved. Investigations on nanoperforated FsNMs (i.e. those with Pdbl ≈ 170 and 65 nm), which are the more regular, reveal that pores crossing the entire membrane thickness do not affect the surface wettability of PLA but drastically enhances the cellular response of this biomaterial. Thus, cell proliferation assays indicate that cell viability in PLA with perforations of Pdbl ≈ 170 nm is ∼2.6 and ∼2.2 higher than in non-perforated PLA and PLA with perforations of Pdbl ≈ 65 nm, respectively. This excellent response has been attributed to the similarity between the nanoperforations with Pdbl ≈ 170 nm and the filopodia filaments in cells (Pdbl ≈ 100-200 nm), which play a crucial role in cell migration processes. The favorable interaction between the perforated membrane nanofeatures and cell filopodia has been corroborated by optical and scanning electron microscopies.