Ascorbate is an abundant and indispensable redox compound in plants. Genetic and biochemical studies have established the d-mannose/l-galactose (d-Man/l-Gal) pathway as the predominant ascorbate biosynthetic pathway in streptophytes, while the d-galacturonate (d-GalUA) pathway is found in prasinophytes and euglenoids. Based on the presence of the complete set of genes encoding enzymes involved in the d-Man/l-Gal pathway and an orthologous gene encoding aldonolactonase (ALase) - a key enzyme for the d-GalUA pathway - Physcomitrium patens may possess both pathways. Here, we have characterized the moss ALase as a functional lactonase and evaluated the ascorbate biosynthesis capability of the two pathways using knockout mutants. Physcomitrium patens expresses two ALase paralogs, namely PpALase1 and PpALase2. Kinetic analyses with recombinant enzymes indicated that PpALase1 is a functional enzyme catalyzing the conversion of l-galactonic acid to the final precursor l-galactono-1,4-lactone and that it also reacts with dehydroascorbate as a substrate. Interestingly, mutants lacking PpALase1 (Δal1) showed 1.2-fold higher total ascorbate content than the wild type, and their dehydroascorbate content was increased by 50% compared with that of the wild type. In contrast, the total ascorbate content of mutants lacking PpVTC2-1 (Δvtc2-1) or PpVTC2-2 (Δvtc2-2), which encode the rate-limiting enzyme GDP-l-Gal phosphorylase in the d-Man/l-Gal pathway, was markedly decreased to 46 and 17%, respectively, compared with that of the wild type. Taken together, the dominant ascorbate biosynthetic pathway in P. patens is the d-Man/l-Gal pathway, not the d-GalUA pathway, and PpALase1 may play a significant role in ascorbate metabolism by facilitating dehydroascorbate degradation rather than ascorbate biosynthesis.