We have de novo designed four antimicrobial peptides AMP‐A/B/C/D, the 51‐residues peptides, which are based on the conserved sequence of cecropin. In the present study, the four peptides were chemically synthesized and their activities assayed. Their secondary structure, amphipathic property, electric field distribution and transmembrane domain were subsequently predicted by bioinformatics tools. Finally, the structure–activity relationship was analyzed from the results of activity experiments and prediction. The results of activity experiments indicated that AMP‐B/C/D clearly possessed excellent broad‐spectrum activity against bacteria, whereas AMP‐A was almost inactive against most of the bacterial strains tested. AMP‐B/C/D showed more potent activity against Gram‐positive bacteria than against Gram‐negative bacteria. By utilizing bioinformatics analysis tools, we found that the secondary structure of the four cation peptides was mainly α‐helix, and the result of CD spectrum also displayed that all the peptides had considerable α‐helix in the presence of either 50% TFE or SDS micelles. AMP‐C showed much better activity than other peptides against most of the bacteria tested, owing to its remarkable cation property and the amphipathic character of its N‐terminal. The study of structure–activity relationship of the designed peptides confirmed that amphipathic structure and high net positive charge were prerequisites for maintaining their activities. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.