Objective To investigate changes in thickness of the skin and muscular layer during the treatment with botulinum toxin A injections in patients with glabellar lines. Methods Twenty-six subjects with moderate to severe glabellar lines were enrolled into this study, who received botulinum toxin A injections in Peking Union Medical College Hospital from August 2017 to February 2018. Before the injections, 4 and 16 weeks after the injections, thickness of the dermis at the injection site was measured by using 50 MHz high-frequency ultrasound, and full thickness of the skin and thickness of the muscular layer were measured with 20 MHz high-frequency ultrasound. Repeated measures analysis of variance, paired t-test and Bonferroni method were used to compare the pretreatment and posttreatment tissue thicknesses measured by using high-frequency ultrasound. Results Before the injections, the thickness of the dermis, full thickness of the skin, thickness of the muscular layer were 2.01 ± 0.48, 4.32 ± 0.73 and 4.51 ± 0.67 mm respectively. No significant difference was observed among the pretreatment, 4- and 16-week posttreatment thickness of the dermis (F = 1.94, P = 0.199) , while there were significant differences among the pretreatment, 4- and 16-week posttreatment full thickness of the skin and thickness of the muscular layer (F = 6.28, 24.19, P = 0.020, 0.016 7) . Conclusions Botulinum toxin A injections can change the thickness of the subcutaneous tissue and muscular layer. High-frequency ultrasound can be applied to the evaluation of efficacy and to follow-up after the treatment of glabellar lines with botulinum toxin A injections. Key words: Ultrasonography; Botulinum toxins, type A; Dermis; Subcutaneous tissue; Muscular layer; Glabellar lines; High-frequency ultrasound; Skin thickness