Steroidal derivatives have attracted tremendous attentions in biological and biomedical areas, due to their variety biological activities. The investigation of structural influences helps in understanding their biological activities. The interactions of steroidal derivatives with DNA may play important roles in biological activities, however only a few investigations were reported on this issue. Herein, the structural influences of three steroidal derivatives were investigated based on their binding interactions with d(GGGT)4 G-quadruplex aptamer by spectroscopic approaches, nuclear magnetic resonance (NMR), electrochemical methods, and molecular modeling techniques. Three compounds were found to selectively bind with parallel G-quadruplex aptamer to form three complexes through end-stacking binding modes. Three compounds stabilized the G-quadruplex structure of the aptamer at different levels, which enhanced the biological activity of this aptamer to some extent. The space steric hindrance was responsible for differences in the binding interactions between d(GGGT)4 G-quadruplex aptamer and three compounds. These results provide new information for the molecular understanding of binding interactions of steroidal derivatives with DNA and the strategy for research of structural influences.