Transcription of the agn43 locus, which specifies an outer membrane protein of Escherichia coli, is regulated in a phase‐variable fashion by the OxyR–DNA binding protein and Dam methylase. Despite its well‐characterized regulation, the function of Ag43 has remained elusive until now. Previous studies indicated that Ag43 mediates autoaggregation of certain strains of E. coli in liquid culture. Given this phenotype, we examined the role of Ag43 in biofilm formation. Here, we report that Ag43 contributes to E. coli biofilm formation in glucose‐minimal medium, but not in Luria–Bertani broth. In addition, we show that flagellar‐mediated motility is required for biofilm formation in both rich and minimal environments. Altogether, our results suggest that E. coli uses both common and specific gene sets for the development of biofilms under various growth conditions.