In situ synchrotron radiation X-ray imaging and diffraction are employed to investigate the deformation behavior of Mg-3Al-1Zn alloy with different twin contents under uniaxial compression. The as-received magnesium alloy is compressed by 4.3% and 9.4% strain to fabricate twins with different contents， the pre-compressed and as-received magnesium alloys are subjected to uniaxial compression under the same condition， and the macroscale stress-strain curves， mesoscale deformation imaging sequence， and microscale X-ray diffraction patterns are obtained simultaneously. The imaging sequences are analyzed via digital image correlation method to resolve deformation fields， and the X-ray diffraction patterns are integrated to identify deformation twins. The results show that abundant ｛1012｝ and ｛1012｝-｛1012｝ twins are activated in the as-received and pre-twined magnesium alloy during uniaxial compression， respectively. Both ｛1012｝ and ｛1012｝-｛1012｝ twins can boost the strain hardening rate by slowing the strain localization process. Compared with the 4.3% pre-compressed sample， more ｛1012｝-｛1012｝ twins are formed in the 9.4% pre-compressed sample， leading to higher efficiency in slowing down strain localization， so that the strain hardening rate and its increase rate are at a high value during uniaxial compression.