Ethambutol (EMB) is a first-line antitubercular drug that inhibits arabinogalactan and lipoarabinomannan biosynthesis. Resistance to EMB has traditionally been associated with embB mutations, especially in the Met306 codon. In this study, the region encompassing the embB306 codon in 109 Mycobacterium tuberculosis clinical isolates (49 EMB-susceptible and 60 EMB-resistant) was characterised. The occurrence of embB306 mutations was verified not only in EMB-resistant isolates (55.0%) but also in EMB-susceptible isolates (16.3%), which questions the role of embB306 mutations as determinants of EMB resistance. Subsequently, four different embB alleles were created by in vitro mutagenesis and were introduced into Mycobacterium smegmatis on a multicopy plasmid. To assess the contribution of embB306 mutations to EMB resistance, the EMB minimum inhibitory concentration (MIC) of these strains was determined. Strains carrying mutant embB306 alleles were able to grow at slightly higher MICs compared with the strain carrying the wild-type embB gene. The small MIC increase obtained here does not appear to be sufficient to cause high-level EMB resistance. The results obtained in the present study suggest that acquisition of EMB resistance might be a multistep process in which embB306 mutations may represent a first-step in EMB resistance acquisition.