A novel fiber-packed column was developed with a fibrous polyimide (PI) as the separation medium in liquid chromatography (LC), and the retention behavior for various aromatic compounds on the stationary phase was investigated in reversed-phase conditions. A retention trend was observed on the PI phase, which strongly retained certain types of analytes with long alkyl chain(s) in the chemical structure. However, the retention factor of analytes with short alkyl chains exceeded that expected from hydrophobic interaction between the analytes and stationary phase. Several solute pairs with and without polar functional groups were employed as the sample probe. The separation factor for these analyte pairs on the PI phase was significantly better than that on typical conventional stationary phases such as octadecylsilica phases. The fibrous PI stationary phase exhibited a unique retention tendency in that the retention time of toluene increased with an increase in the column temperature, and the trends differed from that observed on typical LC stationary phases.