In this paper, the effects of molecular structure on the parameters a, b and c in retention equation are investigated theoretically and experimentally. Parameter b in RP-HPLC approaches a small constant, because there is very weak displacement adsorption. Parameter c in RP-HPLC is mainly determined by the difference between solute-strong solvent and solute-weak solvent interaction and can be quantitatively correlated with the structural parameters of solute such as van der Waals volume (Vw), dipole moment (μA) and hydrogen bond energy (XAH). Parameter a has similar rules to parameter c. The prediction of k’ value for non-polar, homologous compounds is studied. The linear relationship between k’ and coverage of amount of the bonded chain in RP-HPLC is confirmed.