Herein, we report the total and semisyntheses of a series of polymyxin analogues with 2-Thr and 10-Thr modifications to reveal the structure-activity relationship (SAR), which has not been fully elucidated previously. We employed two total-synthetic strategies to facilitate the diversified replacements on 2-Thr or 10-Thr, respectively. Moreover, semisynthetic approaches were utilized to achieve selective esterification of 2-Thr or dual esterification of both 2- and 10-Thr. Based on the results of <i>in vitro</i> antibacterial assays, SAR analysis implicated that the replacement of 2-/10-Thr with amino acids carrying hydrophobic side chains can maintain the activity against <i>Pseudomonas aeruginosa</i> but had varied effects on other tested Gram-negative bacteria. The aminoacetyl esterification on 2-/10-Thr achieved excellent antibacterial activity, and the compound <b>76</b> exhibited 2-8-fold higher activity against different strains and lower toxicity toward the HK-2 cell line. This work explored the SAR of polymyxin 2-/10-Thr and provided a promising strategy for the development of novel polymyxin derivatives.