Objective To investigate the effect of long non-coding RNA Kinectin 1 antisense RNA 1 (lncRNA KTN1-AS1) on the proliferation, migration, and invasion of non-small cell lung cancer (NSCLC) cells and its mechanism of regulating the miR-153-3p/ activated T cell nuclear factor 5 (NFAT5) axis. Methods Real-time quantitative PCR(qRT-PCR) and Western blot were used to measure KTN1-AS1 and miR-153-3p expression and NFAT5 protein expression in NSCLC and adjacent tissues; human normal lung epithelial cells BEAS-2B; and NSCLC cell lines A549, HCC827, and H1299. A549 cells were divided into a Ct group, si-NC group, si-KTN1-AS1 group, mimic NC group, miR-153-3p mimic group, miR-153-3p mimic+pcDNA group, and miR-153-3p mimic+pcDNA-NFAT5 group. qRT-PCR was applied to detect the expressions of KTN1-AS1 and miR-153-3p in cells. CCK-8 assay was applied to detect cell proliferation, and a plate cloning assay was used to detect the ability of cells to form clones. A scratch-healing assay was applied to detect cell migration, and a Transwell assay was used to detect cell invasion. Western blot was applied to detect NFAT5, CyclinD1, matrix metalloproteinase (MMP)-2, and MMP-9 protein expression. Dual luciferase was applied to verify the relationships between KTN1-AS1 and miR-153-3p miR-153-3p, and NFAT5. Results In NSCLC tissues and cells, KTN1-AS1 and NFAT5 proteins were expressed at high levels, and miR-153-3p was expressed at low levels. In A549 cells, KTN1-AS1 and NFAT5 protein expression levels were the highest, and miR-153-3p levels were the lowest (P< 0. 05); therefore, A549 cells were selected as the research subject. Compared with the si-NC group, the si-KTN1-AS1 group showed decreased expression levels of KTN1-AS1 and NFAT5 protein and increased expression levels of miR-153-3p (P< 0. 05). Compared with the mimic NC group, the miR-153-3p mimic group showed statistically comparable KTN1-AS1 expression (P> 0. 05), decreased NFAT5 protein expression, and increased miR-153-3p expression (P< 0. 05). Compared with the miR-153-3p mimic and miR-153-3p mimic + pcDNA groups, the miR-153-3p mimic + pcDNA-NFAT5 group had statistically comparable expression of KTN1-AS1 and miR-153-3p in (P> 0. 05) and increased protein expression of NFAT5 (P< 0. 05). Down-regulation of KTN1-AS1 or up-regulation of miR-153-3p inhibited the proliferation, migration, invasion, and protein expression of CyclinD1, MMP-2, and MMP-9 in A549 cells. Overexpression of NFAT5 attenuated the inhibitory effect of upregulating miR-153-3p on the proliferation, migration, and invasion of A549 cells. KTN1-AS1 targeted the miR-153-3p/ NFAT5 axis. Conclusions Silencing KTN1-AS1 may inhibit the expression of NFAT5 by upregulating miR-153-3p, thereby inhibiting the proliferation, migration, and invasion of A549 cells.