Engineered nanoparticles could trigger inflammatory responses and potentiate a desired innate immune response for efficient immunotherapy. Here we report size-dependent activation of innate immune signaling pathways by gold (Au) nanoparticles. The ultrasmall-size (<10 nm) Au nanoparticles preferentially activate the NLRP3 inflammasome for Caspase-1 maturation and interleukin-1β production, while the larger-size Au nanoparticles (>10 nm) trigger the NF-κB signaling pathway. Ultrasmall (4.5 nm) Au nanoparticles (Au4.5) activate the NLRP3 inflammasome through directly penetrating into cell cytoplasm to promote robust ROS production and target autophagy protein-LC3 (microtubule-associated protein 1-light chain 3) for proteasomal degradation in an endocytic/phagocytic-independent manner. LC3-dependent autophagy is required for inhibiting NLRP3 inflammasome activation and plays a critical role in the negative control of inflammasome activation. Au4.5 nanoparticles promote the degradation of LC3, thus relieving the LC3-mediated inhibition of the NLRP3 inflammasome. Finally, we show that Au4.5 nanoparticles could function as vaccine adjuvants to markedly enhance ovalbumin (OVA)-specific antibody production in an NLRP3-dependent pattern. Our findings have provided molecular insights into size-dependent innate immune signaling activation by cell-penetrating nanoparticles and identified LC3 as a potential regulatory target for efficient immunotherapy.