In vitro, cellular processing on polymeric surfaces is fundamental to the development of biosensors, scaffolds for tissue engineering and transplantation. However, the effect of surface energy and roughness on the cell-surface interaction remains inconclusive, indicating a lack of complete understanding of the phenomenon. Here, we study the effect of surface energy (Es) and roughness ratio (r) of a polydimethylsiloxane (PDMS) substrate on cell attachment, growth, and proliferation. We considered two different cell lines, HeLa and MDA MB 231, and rough PDMS surfaces of different surface energy in the range Es = 21-100 mJ m-2, corresponding to WCA 161°-1°, and roughness ratio in the range r = 1.05-3, corresponding to roughness 5-150 nm. We find that the cell attachment process proceeds through three different stages marked by an increase in the number of attached cells with time (stage I), flattening of cells (stage II), and elongation of cells (III) on the surface. Our study reveals that moderate surface energy (Es ≈ 70 mJ m-2) and intermediate roughness ratio (r ≈ 2) constitute the most favourable conditions for efficient cell adhesion, growth, and proliferation. A theoretical model based on the minimization of the total free energy of the cell-substrate system is presented and is used to predict the spread length of cells that compares well with the corresponding experimental data within 10%. The performance and reusability of the rough PDMS surface of moderate energy and roughness prepared via facile surface modification are compared with standard T-25 cell culture plates for cell growth and proliferation, which shows that the proposed surface is an attractive choice for efficient cell culture. © The Royal Society of Chemistry.