Colorectal carcinoma (CRC) is a common malignant tumor. Increasing evidences indicated that CRC showed a resistance to 5-fluorouracil (5-FU) and further resulted in a poor prognosis. In this study, we aim to investigate the effect of long noncoding RNA nuclear paraspeckle assembly transcript 1 (LncRNA NEAT1) on cell viability, sensitivity to 5-FU, and autophagy of CRC cell lines. MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-Htetrazolium bromide) was used to detect cell viability, immunofluorescent staining was used to detect autophagy puncta, and luciferase reporter system was used to determine binding ability between miR-34a and NEAT1 or putative targets. Additionally, indicated mRNAs and protein expressions were determined by qRT-PCR or western blotting, respectively. We found that NEAT1 expression was increased in CRC tissues and cells, which showed a negative correlation with miR-34a expression. In addition, NEAT1 knockdown noticeably inhibited the proliferation of CRC cells and enhanced 5-FU sensitivity. It revealed that NEAT1 knockdown suppressed the LC3 puncta and the expressions of Beclin-1, ULK1, and ratio of LC3II/I. Overexpression of miR-34a showed similar trends with NEAT1 knockdown. miR-34a was validated to target the putative binding sites in 3'-UTR of HMGB1, ATG9A, and ATG4B, which are involved in the activation of autophagy. Inhibition of miR-34a or overexpression of HMGB1 could effectively reverse elevated 5-FU sensitivity upon NEAT1 knockdown. In addition, 3-MA reversed NEAT1 overexpression-induced resistance in HT29 cells. These findings indicate that LncRNA NEAT1 could target miR-34a and promote autophagy to facilitate 5-FU chemoresistance in CRC. © 2019 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.