We compared ultrasonography (US)-guided injection, targeting the extensor pollicis brevis (EPB) in de Quervain's disease (dQD) with septation, to clinical injection. Forty-four wrists were randomly allocated to US-guided or manual (non-US-guided) injection. At 4 weeks, pain was significantly reduced in both groups. Pain on the 100 mm visual analogue scale (VAS) for the US group was 80.3 (SD 19.6) mm at baseline and 25.6 (SD 15.1) mm at 4 weeks after injection (p = 0.004). Values for the manual group were 78.0 (SD 18.5) mm at baseline and 58.2 (SD 21.9) mm at 4 weeks after injection (p = 0.04). Pain on the VAS showed a more significant decrease in the US-guided than in the manual injection group (p = 0.0007) from baseline to 4 weeks after injection. The results of this study suggest US-guided injection targeting the EPB in dQD patients with septation is more effective than manual injection.