Objective To investigate the effects of BIX-01294, a histone H3 methyltransferase inhibitor, on the viability of protoscoleces and metacestode vesicles of Echinococcus granulosus. Methods Liver samples were collected from E. granulosus-infected sheep in local slaughterhouses. E. granulosus protoscoleces were isolated under sterile conditions and exposed to BIX-01294 solutions (dissolved in DMSO) at concentrations of 1, 5, 10, 25, and 50 µmol/L, while a solvent control group (DMSO group) served as controls. Protoscoleces were collected at 3, 6, 9, 12, and 15 days post-exposure and stained with methylene blue to observe viability. The survival rate of protoscoleces was calculated, and a survival curve was plotted to estimate the area under the survival curve (AUC) was determined using the trapezoidal rule. Protoscoleces were collected following exposure to 25 µmol/L BIX-01294 for 12 days and stained with hematoxylin and eosin (HE) to observe post-exposure changes. Protoscoleces exposed to 1 and 25 µmol/L BIX-01294 for 1, 6, 9, and 12 days were collected and stained with Caspase-3/7 dyes to assess the apoptosis of protoscoleces. The expression of H3K9me2 was determined using Western blotting in protoscoleces exposed to 25 µmol/L BIX-01294 for 12 days. The morphology of vesicles was observed under a microscope following exposure to 25 µmol/L BIX-01294 in vitro for 1, 3, 6, 9, and 12 days, and ultrastructural changes in vesicles were examined using scanning electron microscopy following exposure to BIX-01294 for 9 days. Results The viability of protoscoleces was (36.45 ± 7.14)% on day 6 post-treatment with 25 µmol/L BIX-01294, which significantly reduced as compared with that [(90.21 ± 3.72)%] on day 3 post-treatment (t = 15.86, P < 0.01), and the viability of protoscoleces was (71.91 ± 7.92)% on day 3 post-treatment with 50 µmol/L BIX-01294, which significantly reduced as compared with that (100%) on day 0 post-treatment (t = 6.25, P < 0.01), while the viability was (5.64 ± 1.48)% on day 6 post-treatment with 50 µmol/L BIX-01294, which continued to reduce as compared with that on day 3 post-treatment (t = 15.89, P < 0.01). The AUC values of BIX-01294 decreased in a dose-dependent manner with increasing concentrations, with the lowest AUC seen in the 50 µmol/L BIX-01294 treatment group (395.25%·d). Microscopy displayed shrinkage of protoscoleces, shedding of rostellum and hooklets, reduction of calcium granules and decreased motility following treatment with 50 µmol/L BIX-01294 for 6 days, and HE staining revealed loose, cavitary and irregular protoscoleces with a reduced volume, and thinned and incomplete body wall following treatment with 25 µmol/L BIX-01294 for 12 days. The highest Caspase-3/7 fluorescence intensity was recorded in protoscoleces exposed to 25 µmol/L BIX-01294 for 12 days. Western blotting assay determined lower H3K9me2 expression in protoscoleces treated with BIX-01294 for 12 days than in the DMSO group [(0.53 ± 0.08) vs. (1.00 ± 0.17); t = 4.226, P < 0.05]. Collapse or shrinkage of vesicles was found since day 6 following treatment with 25 µmol/L BIX-01294. Scanning electron microscopy displayed remarkable ultrastructural alterations, separation of germinal layer and cuticle and disorganization of the germinal layer structure in vesicles 9 days post-treatment with BIX-01294. Conclusion BIX-01294 exhibits a remarkable in vitro activity against E. granulosus protoscoleces and vesicles in a time- and concentration-dependent manner, accompanied by reduced H3K9me2 expression and upregulation of apoptosis-related signals, indicating that BIX-01294 has a potential value against echinococcosis.