Abstract Background As a subtype of myosin light chains, myosin light chain 6B (MYL6B) is implicit to cancer development. But its functional characteristic in rectal adenocarcinoma has not been studied determined. Herein, we designed this study to explore the biological significance of MYL6B on rectal adenocarcinoma. Materials and methods To examine the MYL6B expression level, we searched profiles on Oncomine dataset and GEPIA network station. The siRNA strategy was used to knockdown MYL6B, cell counting kit-8 and transwell assays were conducted to measure cell proliferation, migration and invasion respectively. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot were performed to detect the expression level of MYL6B. Results The data showed that overexpress MYL6B was observed in rectal adenocarcinoma tissues and it correlated with a poor prognosis of patients. In vitro function experiments revealed that MYL6B knockdown could inhibit rectal adenocarcinoma cells abilities of cells proliferation, migration and invasion. Moreover, western blot analysis suggested increased E-cadherin level and decreased N-cadherin and Vimentin expressions after knockdown MYL6B. Conclusion Collectively, this study elaborated the promoting effect of MYL6B in rectal adenocarcinoma progression, thus providing novel insight for strategies of clinical diagnosis and drug application in the future clinical study.