Long-acting (lasting extend) diquafosol ophthalmic solution 3% (DQSLX) is administered three times daily versus six times daily for the currently approved diquafosol ophthalmic solution (DQS). We investigated the efficacy and adherence of switching from DQS to DQSLX in patients with dry eye disease. We retrospectively enrolled 54 patients (17 men and 37 women) with eye drop prescription changes from DQS to DQSLX between December 2022 and March 2023. The number of eye drops, subjective symptoms, tear breakup time (TBUT), and fluorescein staining scores from baseline to 4 weeks after starting DQSLX were evaluated. Participants then chose between DQSLX and DQS. Patients administered DQSLX three times per day, as listed on the package insert, 88.9% of the time; significantly higher than the 5.6% of patients who used DQS six times per day, as instructed. The DQSLX group showed significant improvements in symptoms and fluorescein staining scores (23.3 ± 20.1 and 0.8 ± 1.7, respectively) compared with the baseline (37.8 ± 24.1 and 1.1 ± 1.5,p =0.01 and <0.001, respectively). The TBUT in the DQSLX group (5.0 ± 2.5 s) did not significantly improve compared to the DQS group (4.5 ± 1.7 s) (p =0.75). Fifty-one (94.4%) patients opted to continue DQSLX because of the pleasant feeling of the eye drops, long-lasting moisture, and less frequent administration. The efficacy and adherence of DQSLX was comparable to DQS.