The interaction of cytochrome c (cyt c) with poly(isobutylene-alt-maleic acid) (PIMA) and poly(1-tetradecenealtmaleic acid) (PTMA) was studied using circular dichroism, absorption spectroscopy, and atomic force microscopy to investigate the electrostatic and hydrophobic influence of the copolymers on the structure of cyt c. At pH 7.4, the interaction of PIMA with cyt c can only partly disturb the integrity of the heme pocket, while PTMA has very intensive influence on the structure of cyt c. After adding 0.15 M NaCl, PIMA-cyt c complexes dissociate, and the released cyt c recovers its native structure, whereas NaCl has no significant influence on PTMA-cyt c complexes. GuHCl (0.5 M) destroys PTMA-cyt c complexes, forming GuHCl-PTMA precipitates; the cyt c released from the complexes regenerates its native structure. In comparison with electrostatic interaction, hydrophobic interaction leads to more stable polymer-cyt c complexes and more intensive influence on cyt c structure, but cyt c can recover its native state after release.