Charged substrates are expected to promote cell adhesion via electrostatic interaction, but it remains unclear how cells adhere to these substrates. Here, initial cell adhesion (<30 min) was re-examined on charged substrates in serum-containing and serum-free media to distinguish among various cell adhesion mechanisms (i.e., electrostatic interaction, hydrophobic interaction, and biological interaction). Cationic and anionic methacrylate copolymers were coated on nonionic nontissue culture-treated polystyrene to create charged substrates. Cells adhered similarly on cationic, anionic, and nonionic substrates in serum-free medium via integrin-independent mechanisms, but their adhesion forces differed (anionic > cationic > nonionic substrates), indicating that cell adhesion is not mediated solely by the cells' negative charge. In serum-containing medium, the cells adhered minimally on anionic and nonionic substrates, but they adhered abundantly on cationic substrates via both integrin-dependent and-independent mechanisms. These results suggest that neither electrostatic force nor protein adsorption is accountable for cell adhesion. Conclusively, the observed phenomena revealed a gap in the generally accepted understanding of cell adhesion mechanisms on charged polymeric substrates. A reanalysis of their mechanisms is necessary.