Introduction and Objective: Alveolar osteoblasts have critical functions during alveolar bone regeneration. Berberine (BBR) and microRNAs (miRNAs) are considered to play important roles in regulating osteoblast differentiation. The study aimed to investigate the role and mechanisms of BBR in osteogenic differentiation of human alveolar osteoblasts (HAOBs) and determine miR-214 expression in the process. Methods: Healthy human alveolar bones were cultured in vitro and prepared for morphological observation and alkaline phosphatase (ALP) staining. The third generation of HAOBs was used for cell transfection and treated by different concentrations of BBR. Cell Counting Kit-8 was used to detect the effect of BBR and increased miR-214 on the proliferation of HAOBs. qRT-PCR and Western blot were used to detect the expression of osteogenic differentiation-related genes and miR-214 level, respectively. Results: The ALP staining results were positive, indicating that cultured cells were HAOBs. Different concentrations of BBR significantly promoted the proliferation of HAOBs and increased the expression levels of ALP, osteocalcin (OCN), collagen type I alpha 1 (COL1A1), runt related transcription factor 2 (RUNX2), and osterix (OSX). Moreover, the expression of miR-214 was reduced as BBR concentrations increased, and the increase of miR-214 reversed the BBR-induced proliferation and osteogenic differentiation of HAOBs. Conclusion: BBR could promote the proliferation and osteogenic differentiation of HAOBs through downregulating the expression of miR-214.