Three new β-class milbemycins, 13α-hydroxy-4-ethy1 milbemycin β3 (1), 13α-hydroxy-25-ethy1 milbemycin β3 (2), 13α-hydroxy milbemycin β3 (3), were isolated from the broth of the genetically engineered strains Streptomyces avermitilis MHJ1011, whose aveA1 gene was replaced by milA1 gene seamlessly. Their structures were determined on the basis of extensive spectroscopic analysis and comparison with data from the literature. These three compounds, especially compound 1, exhibited potent acaricidal activity.