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Recently, a putative alcohol dehydrogenase 3, termed EhADH3B of the <i>Entamoeba histolytica</i> isolate HM-1:IMSS was identified, which is expressed at higher levels in non-pathogenic than in pathogenic amoebae and whose overexpression reduces the virulence of pathogenic amoebae. In an <i>in silico</i> analysis performed in this study, we assigned EhADH3B to a four-member ADH3 family, with <i>ehadh3b</i> present as a duplicate (<i>ehadh3b^a</i>/<i>ehadh3b^b</i>). In long-term laboratory cultures a mutation was identified at position 496 of <i>ehadh3b^a</i>, which codes for a stop codon, which was not the case for amoebae isolated from human stool samples. When using transfectants that overexpress or silence <i>ehadh3b^b</i>, we found no or little effect on growth, size, erythrophagocytosis, motility, hemolytic or cysteine peptidase activity. Biochemical characterization of the recombinant EhADH3B^b revealed that this protein forms a dimer containing Ni²⁺ or Zn²⁺ as a co-factor and that the enzyme converts acetaldehyde and formaldehyde in the presence of NADPH. A catalytic activity based on alcohols as substrates was not detected. Based on the results, we postulate that EhADH3B^b can reduce free acetaldehyde released by hydrolysis from bifunctional acetaldehyde/alcohol dehydrogenase-bound thiohemiacetal and that it is involved in detoxification of toxic aldehydes produced by the host or the gut microbiota.