Bacterial persistence is one of the major causes of antibiotic treatment failure and the step stone for antibiotic resistance. However, the mechanism by which persisters arise has not been well understood. Maintaining a dormant state to prevent antibiotics from taking effect is believed to be the fundamental mechanistic basis, and persisters normally maintain an intact cellular structure. Here we examined the morphologies of persisters in Acinetobacter baumannii survived from the treatment by three major classes of antibiotics (i.e. β-lactam, aminoglycoside, and fluoroquinolone) with microcopy and found that a fraction of enlarged spherical bacteria constitutes a major sub-population of bacterial survivors from β-lactam antibiotic treatment, whereas survivors from the treatment of aminoglycoside and fluoroquinolone were less changed morphologically. Further studies showed that these spherical bacteria had completely lost their cell wall structures but could survive without any osmoprotective reagent. The spherical bacteria were not the viable-but-non-culturable cells and they could revive upon the removal of β-lactam antibiotics. Importantly, these non-walled spherical bacteria also persisted during antibiotic therapy in vivo using Galleria mellonella as the infection model. Additionally, the combinational treatment on A. baumannii by β-lactam and membrane-targeting antibiotic significantly enhanced the killing efficacy. Our results indicate that in addition to the dormant, structure intact persisters, the non-wall spherical bacterium is another important type of persister in A. baumannii. The finding suggests that targeting the bacterial cell membrane during β-lactam chemotherapy could enhance therapeutic efficacy on A. baumannii infection, which might also help to reduce the resistance development of A. baumannii.