ObjectiveThis study aimed to explore the role of miR‐140‐5p in cranial base synchondrosis chondrocytes (CBSCs) under cyclic tensile strain (CTS).Setting and Sample PopulationA total of 25 1‐week‐old Sprague Dawley rats from Shanghai Laboratory Animal Center, Chinese Academy of Sciences, were used.Material and MethodsThe second passage of CBSCs was applied with CTS at 10% elongation (1 Hz) for 24 hours. MiR‐140‐5p levels in CBSCs were detected by qRT‐PCR. The role of miR‐140‐5p in CBSCs was evaluated by transfection of mimics and inhibitor. RNA sequencing and online search of miRNA databases (TargetScan, miRDB and miRanda) were used in prediction of miR‐140‐5p targets. A luciferase reporter assay was applied to identify the target gene of miR‐140‐5p.ResultsCompared with the control, the expression of Col2a1 and Sox9 was significantly higher after CTS (P < .05). Also, CBSCs demonstrated higher expression of miR‐140‐5p after CTS loading for 24 hours (P < .05). Overexpression of miR‐140‐5p promoted ECM synthesis under CTS loading environment, while suppression of miR‐140‐5p inhibited the effect. Bloc1s2 was a putative target gene of miR‐140‐5p.ConclusionsThe expression of ECM in CBSCs could be promoted by CTS and miR‐140‐5p might play a role in this process through targeting Bloc1s2.