This paper reports the initial process of the enzymatic degradation of solution-grown lamellar single crystals of bacterial poly[(R)-3-hydroxybutyrate] (P(3HB)) with an extracellular polyhydroxybutyrate (PHB) depolymerase purified from Alcaligenes faecalis T1. We used a hydrolytic-activity-disrupted mutant of the PHB depolymerase in order to avoid the influence of hydrolytic reaction in the system. The effect of addition of the mutant enzyme upon the P(3HB) single crystals was investigated by turbidimetric assay, high-performance liquid chromatography (HPLC), and atomic force microscopy (AFM). Suspension turbidity of the P(3HB) single crystals increased after addition of the mutant enzyme having no hydrolytic activity. No soluble product from the P(3HB) single crystals with the mutant enzyme was detected by HPLC. AFM observation of the P(3HB) single crystals adsorbed on highly ordered pyrolytic graphite revealed that the mutant enzyme yielded a lot of lengthwise crystal fragments from the P(3HB) single crystals. On the basis of these results, we concluded that the mutant enzyme disturbs the molecular packing of the P(3HB) polymer chain around the loose chain packing region in the single crystal, resulting in the fragmentation. Therefore, it is suggested that the enzymatic degradation of P(3HB) single crystals with a wild-type PHB depolymerase progresses via three steps: (1) adsorption of the enzyme onto the surface of the single crystal; (2) disturbance of the molecular packing of P(3HB) polymer chain in the single crystal by the adsorbed enzyme; and (3) hydrolysis of the disturbed polymer chain by the adsorbed enzyme.