The side chain of a compound plays an important role in its biological function. In our studies, we have found that hydroximinosteroid derivatives with different side chains and position of hydroximino on ring A and B displayed remarkable distinct cytotoxicities against a diversity of cancer cell types. Presence of an oxime group on ring B and a hydroxy on ring A or B resulted in a higher cytotoxicity than other structural motifs. In addition, a cholesterol-type side chain at position 17 was required for the biological activity. Our findings provide new evidence showing the relationship between the chemical structure and biological function. The information obtained from the studies may be useful for the design of novel chemotherapeutic drugs.