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Abstract Study design Retrospective study. Objective This study aimed to compare the fusion order between the cranial and caudal levels in two-level anterior cervical discectomy and fusion (ACDF) with a zero-profile device in the treatment of cervical spondylopathy. Summary of background data Fusion is the standard used to judge the success of ACDF. However, the fusion order in two-level ACDF remains uncertain. The mechanical environment of different levels is different, which may affect the fusion rate or fusion order. Methods From 2014 to January 2019, data of consecutive patients with two-level cervical disk degenerative disease who underwent ACDF were retrospectively reviewed. Radiological assessments were based on the range of motion of the fusion level, segment slope, and disk height, and complications were assessed. Data were analyzed using the paired t, Mann-Whitney U, χ 2, Fisher exact, and rank-sum tests and logistic regression analysis. Results In total, 118 patients were ultimately enrolled for analysis in the study. The respective fusion rates of the cranial and caudal levels were 26.27% and 10.17% (p < 0.05) at 3 months, 58.47% and 42.37% (p < 0.05) at 6 months, 86.44% and 82.20% (1 0.05) at 1 year, and 92.37% and 89.83% (p > 0.05) at the last follow-up. Multivariate logistic regression analysis indicated that the preoperative segmental slope and cranial level were independent risk factors for non-fusion. The adjacent segment degeneration (ASD) and subsidence rates were comparable between the two levels. Conclusion The caudal level had a slower fusion process than the cranial level. A higher preoperative segment slope was a risk factor for fusion. However, the subsidence and ASD rate were comparable between the caudal and cranial levels in the two-level ACDF.