AbstractBackgroundHuman umbilical cord mesenchymal stem cells (hUC‐MSCs) have the ability to differentiate into nerve cells, which offers promising options for treating neurodegenerative diseases.AimTo explore the important regulatory molecules of hUC‐MSCs differentiation into neurons.MethodIn this research, the neural differentiation of hUC‐MSCs was induced by a low‐serum DMSO/BHA/DMEM medium. The GEO database was used to retrieve the relevant datasets. The starBase and miEAA databases were used for bioinformatics analysis. RT‐qPCR was used to detect the hsa‐miR‐543 level and the mRNA levels of NSE, NeuN, NF‐M, KIF5C, and CALM3. The protein levels of KIF5C and CALM3 were checked by western blotting.ResultsThe expression levels of NSE, NeuN, NF‐M, KIF5C, and CALM3 were elevated, while hsa‐miR‐543 was under‐expressed in neuro‐induced hUC‐MSCs. The increase in NSE, NeuN, and NF‐M mRNA levels induced by DMSO/BHA/DMEM was partially reversed by the knockdown of KIF5C and CALM3 in hUC‐MSCs. Moreover, the transfection of hsa‐miR‐543 mimic partially countered the DMSO/BHA/DMEM‐induced elevation in NSE, NeuN, NF‐M, KIF5C, and CALM3 mRNA levels.ConclusionKIF5C and CALM3 facilitated the neuronal differentiation of hUC‐MSCs, whereas hsa‐miR‐543 exerted an opposing effect by negatively regulating KIF5C and CALM3.