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Bifidobacteria have been investigated due to their mutualistic microbe–host interaction with humans throughout their life. This work aims to make a biochemical and genomic characterization of <i>Bifidobacterium pseudocatenulatum</i> JCLA3. By multilocus analysis, the species of <i>B. pseudocatenulatum</i> JCLA3 was established as <i>pseudocatenulatum</i>. It contains one circular genome of 2,369,863 bp with G + C content of 56.6%, no plasmids, 1937 CDSs, 54 tRNAs, 16 rRNAs, 1 tmRNA, 1 CRISPR region, and 401 operons predicted, including a CRISPR-Cas operon; it encodes an extensive number of enzymes, which allows it to utilize different carbohydrates. The <i>ack</i> gene was found as part of an operon formed by <i>xfp</i> and <i>pta</i> genes. Two genes of <i>ldh</i> were found at different positions. Chromosomally encoded resistance to ampicillin and cephalothin, non-hemolytic activity, and moderate inhibition of <i>Escherichia coli</i> ATCC 25922 and <i>Staphylococcus aureus</i> ATCC 6538 were demonstrated by <i>B. pseudocatenulatum</i> JCLA3; it can survive 100% in simulated saliva, can tolerate primary and secondary glyco- or tauro-conjugated bile salts but not in a mix of bile; the strain did not survive at pH 1.5–5. The <i>cbh</i> gene coding to choloylglycine hydrolase was identified in its genome, which could be related to the ability to deconjugate secondary bile salts. Intact cells showed twice as much antioxidant activity than debris. <i>B. pseudocatenulatum</i> JCLA3 showed 49% of adhesion to Caco-2 cells. The genome and biochemical analysis help to elucidate further possible biotechnological applications of <i>B. pseudocatenulatum</i> JCLA3.