As alternatives to perfluorooctanoic acid (PFOA), hexafluoropropylene oxide (HFPO) homologues, including hexafluoropropylene oxide dimer acid (HFPO-DA), hexafluoropropylene oxide trimer acid (HFPO-TA), and hexafluoropropylene oxide tetramer acid (HFPO-TeA), have been used in the fluoropolymer industry for a long period of time. These compounds have attracted widespread attention in recent years due to their environmental ubiquity and high bioaccumulation capability, as well as their toxicity. In our study, we evaluated the potential estrogenic effects of HFPOs in comparison to PFOA by ligand binding, transcriptional activity, and in vivo assays. Fluorescence ligand binding assays showed that both HFPO-TA and HFPO-TeA exhibited higher binding affinity to estrogen receptor ligand binding domains (ER-LBDs) than PFOA, with 2.5- and 57.5-fold higher affinity to ERα-LBD and 2.6- and 41.8-fold higher affinity to ERβ-LBD, respectively, whereas HFPO-DA exhibited weaker binding affinity than PFOA. Unlike PFOA, HFPO-TA and HFPO-TeA exhibited antagonistic activity toward the ERs' signaling pathway, with HFPO-TeA displaying the strongest potency. In silico study revealed that while PFOA binds with ERs in a similar fashion as 17β-estradiol, the HFPOs display an antagonistic binding mode. Using a zebrafish model, we further found that exposure to HFPO homologues significantly altered the levels of sex steroid hormones and vitellogenin. In general, both in vivo and in vitro results indicate that HFPO homologues might exert higher estrogenic effects than PFOA.