Abstract <p>The inactivated vaccines have played a pivotal role in the control and eradication of foot-and-mouth disease (FMD). However, certain safety concerns remain. Recently, virus-like particles (VLPs) have gradually become a research hotspot. As the eukaryotic expression system with the lowest production costs, the production of VLPs using <i>Pichia pastoris</i> has significant potential. During the natural infection process of FMD virus (FMDV), the polyprotein P1 is cleaved by 3C protease to form VP0, VP3, and VP1, which are subsequently assembled into VLPs. In this study, we adopted an alternative approach, co-expressing VP0, VP3, and VP1 without 3C protease for the production of FMDV VLPs in <i>P. pastoris</i>. The western blot (WB) assays showed variable protein expression on the same plasmid. VP0 was the highest, while VP3 and VP1 were similar. Furthermore, the order of proteins on the plasmid also mattered. The results indicated that His₆ tags at VP0, VP3, and VP1 N-termini significantly affected VLPs assembly. The three-dimensional structure of FMDV revealed that the N-terminus of VP3 and VP1, which are situated in the external space of VLPs, can be fused with His₆ tag. Inserting His₆ tags into the G-H loop region of VP1 did not hinder assembly, thus providing a reference for the affinity purification of capsid and VLPs assembly. Here, FMDV VLPs were successfully produced independently of 3C protease, avoiding the uncontrollable cleavage efficiency and toxicity of 3C protease in host cells and demonstrating the potential of <i>P. pastoris</i> for FMDV VLPs production.</p> Key points <p><i>• FMDV VLPs could be produced in P. pastoris by a 3 C protease–independent approach</i></p> <p><i>• Optimal expression of FMDV VLPs in P. pastoris is achieved at pH 7 with 72-h induction</i></p> <p><i>• His</i>_<i>6</i><i> can be fused to the G-H region and C-terminus of VP1 and C-terminus of VP3 without affecting the VLPs assembly</i></p>