β-D-Galactosidase (EC 3.2.1.23) has been detected in several plant species, and is characterized in different organs and tissues by its ability to hydrolyse terminal non-reducing β-D-galactosyl residues from β-D-galactoside polymers. In the present paper the cloning and the biochemical and molecular characterization of Coffea arabica β-galactosidase expressed in the pericarp and the endosperm of coffee fruits in all phases of ripeness are described. It was found that coffee β-galactosidase is not evenly transcribed throughout fruit ripening, oscillating with two distinct peaks: the first peak when immature fruits are at the active growing stage and the second when fully developed coffee fruits are completely ripe. Both in vitro enzymatic activity of coffee fruit protein extracts and in vivo histochemical assay of freshly harvested coffee fruits confirmed the uneven transcription of β-galactosidase as fruit maturation advanced. Partial genomic DNA sequencing indicated a complex arrangement of nine putative exons. In silico translation of the cloned coding sequences clearly revealed the cloned gene as β-galactosidase, with the presence of a signal peptide directing the enzyme to the apoplast. Two isoforms were distinguished by sequencing reverse transcription-PCR transcripts, one expressed in young and adult leaves and another in stems, petals, and coffee fruit endosperm and pericarp. Southern blot analysis indicates that there are at least two copies of this gene in the C. arabica genome that could explain the presence of two β-galactosidase isoforms.