The physicochemical properties of nanoparticles have been tuned via various synthetic methods to improve their diagnostic or curative capability. However, systematic understanding of the relationship between their physicochemical properties and biological effects is still not well established. Particularly, the latent ability of nanomaterials to regulate autophagy has already drawn more attention. In this report, by comparing cellular interactions, uptakes, and autophagic effects of gold nanoparticles with different shapes, we reveal that gold nanoparticles could modulate autophagy in a shape-dependent manner. Western blot assays and confocal images confirm that nanospheres cause more autophagosome accumulation than nanorods, which are highly correlated with the difference in cellular uptakes. With biological TEM, we observe remarkable lysosome swelling and clearly identify the engulfed gold nanoparticles together with undegraded organelles in autolysosomes. Additionally, monitoring of the lysosomal activity and p62 degradation indicates an autophagy flux decrease induced by the impairment of lysosomes after treatment with nanoparticles. Our study not only reveals the effects of nanostructure morphology on autophagy, but also provides an alternative strategy to modulate autophagy, which would contribute to the guidelines for further biomedical applications of various nanomaterials.