The main chain stiffness parameter (lambda M-1) of cylindrical rod brushes consisting of a flexible polymethacrylate (PMA) main chain and poly(n-hexyl isocyanate) (PHIC) rod side chains has been determined in tetrahydrofuran (THF) at 25 degrees C by static light and small-angle X-ray scattering measurements. Furthermore, we have rationalized lambda M-1 as a function of the degree of polymerization of HIC, N-s. The lambda(-1)(M) values were compared to those of rod brushes consisting of a flexible polystyrene (PSt) main chain [Macromolecules 2008, 41, 6564] to reveal how the molecular conformation of the rod brush is influenced by the difference in the primary structure or chemical nature of the main chain. A series of rod brushes, poly(MA-HIC-N-s-Ac) with different side chain lengths, where N, ranged from 26 to 82, were prepared by radical homopolymerization of a-methacryloyloxyethoxy-omega-acetyl ended PHIC macromonomers (MA-HIC-N-s-Ac) in n-hexane at 60 degrees C. The mean-square cross-section radius of gyration ((S-c(2))) of poly(MA-HIC-N-s-Ac) was essentially consistent with those of the rod brushes consisting of a PSt main chain, poly(VB-HIC-Ns-H), and could be quantitatively described by the wormlike comb model. The z-averaged mean-square radius of gyration ((< S >(2))) of the brush, determined as the function of molecular weight, was analyzed with the aid of the cylindrical wormlike chain model theory to determine the lambda(-1)(M) value. The scattering form factor of poly(MA-HIC-Ns-Ac) was also quantitatively described by the wormlike chain model, and the corresponding 4,lambda(-1)(M) parameter was determined. The lambda(-1)(M) value of poly(MA-HIC-N,-AO was much greater than that of poly(VB-HIC-Ns-H) having the same N, and increased in proportion with increasing side chain length (lambda(-1)(M) proportional to N-s-H). The chemical nature of the main chain, which is the minor component in the brush polymers, was found to play an essential role in the overall conformation of the rod brushes.