Lactoferrin and polyphenols are common natural functional compounds. Their interactions and the consequential alterations in functional activity have received widespread attention. The work aimed to investigate the interaction between lactoferrin and kaempferol, as well as evaluate the in vitro anticancer properties of the lactoferrin–kaempferol complex. The results of the spectra experiments revealed that lactoferrin and kaempferol are capable of forming complexes to quench the endogenous fluorescence of lactoferrin. Further insight into the binding mechanism was gained through molecular docking and molecular dynamics simulations. These analyses suggest that both hydrophobic interactions and hydrogen bonding are essential factors in the interaction between lactoferrin and kaempferol. Furthermore, the MTT assay and apoptosis by flow cytometry were conducted, revealing a synergistic effect of kaempferol and lactoferrin on the inhibition of HeLa cell proliferation. The findings from this investigation could improve our understanding of lactoferrin's interaction with polyphenols and the role of the lactoferrin–kaempferol complex in inhibiting cancer cell proliferation.