α-Phase poly(vinylidene fluoride) (PVDF) has chains of zero dipole moments and is, therefore, nonpiezoelectric, while β-phase PVDF has the most significant piezoelectric properties among the polymorphs due to its polar chains. Although many reports describe PVDF as a suitable biomaterial due to its stability and biocompatibility, few considered the specific effects that the different polymorphs exert on cellular behaviour. We hypothesized that α- and β-phase PVDF will exert direct but different influences on cell attachment and metabolic activity. PVDF films were fabricated using N,N-dimethylformamide (DMF) and hexamethylphosphoramide (HMPA) by solvent casting. Samples were characterized by differential scanning calorimetry, Fourier transform infrared spectroscopy and X-ray diffraction. Films containing 83.5% α-phase PVDF (DMF-PVDFα) and 91.4% of β-phase PVDF (HMPA-PVDFβ within the crystalline regions were produced and used to evaluate in vitro attachment and metabolic activity of L929 cells. Cell metabolic activity on both PVDF conformations increased 3-fold over the 1-week culture period, with higher cell metabolic activity observed on DMF-PVDFα on day 5 of culture, compared to HMPA-PVDFβ. Cells grown on DMF-PVDFα were well-spread, flat and expressed spotted paxillin in focal adhesions that were mainly localized to perinuclear regions of the cells, while a high proportion of cells on HMPA-PVDFβ were bulging, round and expressed relatively fewer paxillin spots. Our results suggest that α-phase PVDF supports higher cell metabolic activity and better cell spreading compared to β-phase PVDF. Such variations can potentially be exploited for different biomedical applications.