Although the pellicle is one of the major growth modes of microorganisms, the metabolic features of pellicle cells and the determinative factors for pellicle formation are largely unknown. In recent years, biofilm development of Shewanella oneidensis, an important model organism for bioremediation studies, has been extensively studied. In this paper, a transcriptional profiling of pellicle cells relative to planktonic cells indicated that cells in pellicles were more metabolically active than the planktonic cells. Most notably, up-transcription of general secretion system proteins and iron/heme uptake and transport proteins was observed in pellicle cells. Unexpectedly, neither the hmuT nor hugA heme transport mutant exhibited a significant defect in pellicle formation. Expectedly, three type I secretion system mutants were severely deficient in pellicle formation, suggesting an essential role of these proteins.