Highlights ► The effects of butenolide on animal and human embryonic development are unknown. ► The embryotoxicity of butenolide is studied via an whole embryo culture model. ► Butenolide induces growth retardation and differentiation inhibition. ► The viability of 3T3-cell is impaired at 2.5mg/L, and the IC50 3T3 is 6.45mg/L. ► Butenolide exerts detrimental effects on rat embryo development in vitro.