Purpose: This study aimed to use proteomics methods to investigate the changes in serum protein levels after high- and low-flux hemodialysis (HD). Methods: Before and after HD, serum samples were obtained from two selected patients who were treated with a Polyflux 140 H high-flux dialyzer and a Polyflux 14 L low-flux dialyzer during two continuous therapy sessions. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to identify the proteins. Results: A total of 212 and 203 serum proteins were identified after high-flux and low-flux HD, respectively. After high-flux HD, 21 proteins increased, and 132 proteins decreased. After low-flux HD, 87 proteins increased, and 45 proteins decreased. High-flux HD led to a significantly greater reduction in protein levels than low-flux HD (0.73 ± 0.13 vs. 0.84 ± 0.18, p = .00). Among the increased and decreased proteins, the isoelectric point (pI) values mainly ranged from 5 to 7, and the molecular weights (Mws) were mostly smaller than 30 kDa. The serum proteins showed no difference in pI or Mw for high- and low-flux HD. Gene ontology (GO) analysis showed that the detected proteins were related to immune system processes and complement activation. Conclusions: Serum protein levels differentially changed after high- and low-flux HD. Long-term effects should be observed in future studies.