Aim Bacteria naturally produce membrane vesicles (MVs), which have been shown to contribute to the spread of multi‐drug resistant bacteria (MDR) by delivering antibiotic‐resistant substances to antibiotic‐susceptible bacteria. Here, we aim to show that MVs from Gram‐positive bacteria are capable of transferring β‐lactam antibiotic‐resistant substances to antibiotic‐sensitive Gram‐negative bacteria. Materials and Methods MVs were collected from a methicillin‐resistant strain of Staphylococcus aureus (MRSA) and vesicle‐mediated fusion with antimicrobial‐sensitive Escherichia coli (RC85). It was performed by exposing the bacteria to the MVs to develop antimicrobial‐resistant E. coli (RC85‐T). Results The RC85‐T exhibited a higher resistance to β‐lactam antibiotics compared to the parent strain. Although the secretion rates of the MVs from RC85‐T and the parent strain were nearly equal, the β‐lactamase activity of the MVs from RC85‐T was 12‐times higher than that of MVs from the parent strain, based on equivalent protein concentrations. Moreover, MVs secreted by RC85‐T were able to protect β‐lactam‐susceptible E. coli from β‐lactam antibiotic‐induced growth inhibition in a dose‐dependent manner. Conclusion MVs play a role in transferring substances from Gram‐positive to Gram‐negative bacteria, shown by the release of MVs from RC85‐T that were able to protect β‐lactam‐susceptible bacteria from β‐lactam antibiotics. Significance and impact of study MVs are involved in the emergence of antibiotic‐resistant strains in a mixed bacterial culture, helping us to understand how the spread of multidrug‐resistant bacteria could be reduced.