To identify the optimal method for endometrial preparation in vitrified single-blastocyst transfer (VSBT) cycles. This was a retrospective cohort study for non-PCOS patients who underwent VSBT cycles from March 2015 to November 2019 in an academic reproductive medical center. A total of 6840 VSBT cycles were enrolled and classified into two groups according to different endometrial preparation methods. The non-PCOS patients who underwent VSBT showed a significantly higher clinical pregnancy rate (61.96% vs 56.85%, p < 0.001) and live birth rate (49.09% vs 39.86%, p < 0.001), as well as a statistically lower early miscarriage rate (12.02% vs 18.08%, p < 0.001) in the natural cycle (NC) group compared with the artificial cycle (AC) group. Multivariable analysis further confirmed that NC was associated with an increased likelihood of clinical pregnancy (adjusted odds ratio(aOR) 0.852, 95% confidence interval (CI) 0.765-0.949, p = 0.004) and live birth (aOR0.746, 95% CI 0.669-0.832, p < 0 .001), but decreased early miscarriage occurrence (aOR 1.447, 95% CI 1.215-1.724, p < 0.001) compared to AC. Our study demonstrated that non-PCOS patients could benefit from NC in vitrified blastocyst transfer. Increased clinical pregnancy rate and decreased early miscarriage rate led to a significantly higher live birth rate in NC patients compared with AC with our present protocol.