Autophagy, an evolutionarily conserved cellular self-digestive process, is associated with different diseases and can be inhibited or induced by a series of agents. In this study, we reported that fangchinoline (FCL), an alkaloid from Stephania tetrandra S. Moore, increased the expression of LC3-II and the formation of GFPLC3 puncta in non-small cell lung cancer (NSCLC) cells. Numerous yellow puncta were observed in mRFPEGFP- LC3 stably expressed NSCLC cells under FCL treatment and the FCL-increased expression of LC3-II was not further increased after co-treatment with bafilomycin A1, an autophagy inhibitor, suggesting that FCL inhibits autophagic flux. Results of co-localization of GFP-LC3 with LysoTracker Red or lysosomal-associated membrane protein 1 indicated that FCL inhibits autophagosomes-lysosomes fusion. Furthermore, FCL decreased the activities of cathepsin B and cathepsin D and affected the cellular acidification. Interestingly, FCL also increased the nuclear translocation of transcription factor EB (TFEB), a master regulator of autophagic and lysosomal genes, and the mRNA expressions of TFEB-targeted genes, such as SQSTM1, MAP1LC3B, and UVRAG. Knockdown of TFEB by using small inference RNA decreased the FCL-induced expression of LC3-II and the formation of GFP-LC3 puncta. Overall, we reported that FCL increases the expressions of autophagy markers via both inhibition (inhibition of autophagosomes-lysosomes fusion and dysfunction of lysosome) and induction (promotion of TFEB nuclear translocation) of autophagy, providing insights into the better understanding of the complexity of agent-mediated autophagy.