The aim of this study was to perform neural differentiation of mesenchymal stem cells derived from Wharton’s jelly of human umbilical cord (HUMSCs) and explore the role of miR-20b and miR-106a, which may regulate Neurogenin-2 (Ngn2) expression during the neural differentiation. HUMSCs were cultured and induced in vitro. The cells were stained for nestin and microtubule-associated protein 2 (MAP2) by immunofluorescence. The interactional binding sites between the 3′-untranslated region (3′-UTR) of Ngn2 mRNA and miR-20b or miR-106a were predicted by bioinformatics and identified by a dual-luciferase assay. The expressions of Ngn2, miR-20b, and miR-106a were determined by real-time PCR and western blot before and after the neural differentiation. After infection of miR-20b or miR-106a, the expressions of Ngn2, MAP2, and β III-tubulin (TUBB3) were measured. HUMSCs showed a uniform pattern with a typical short spindle-shaped morphology. Fourteen days after neural differentiation, HUMSCs showed neuronal traits of pyramidal appearance. TargetScan and miRanda showed that miR-20b and miR-106a were well complementary with Ngn2 3′-UTR. Identified by the dual-luciferase assay, we found that miR-20b and miR-106a inhibit Ngn2 expression by binding to its 3′-UTR. Furthermore, the expression of Ngn2 mRNA was almost reciprocal to that of miR-20b and miR-106a by real-time PCR during the neural differentiation of HUMSCs. Overexpression of miR-20b and miR-106a downregulated the expressions of Ngn2, MAP2, and TUBB3. miR-20b and miR-106a may directly or indirectly regulate neuronal genes expression to modulate the neural differentiation of HUMSCs.