Vector vaccines based on human adenovirus type 26 (HAdV-D26) have been intensively researched for several years. Regardless, the mechanisms of activation of the innate immune system by this virus have not yet been fully investigated. The inflammasome is a signaling complex that via caspase-1 activates pro-inflammatory interleukins important for the inflammatory response and protection against pathogens. The aim of this study was to investigate how inhibition of clathrin-mediated endocytosis affects inflammasome activation in human epithelial cells infected with HAdV-D26. The quantitative polymerase chain reaction revealed a decrease in gene expression for pro-inflammatory interleukins IL-1β i IL-18 and caspase-1 after infection, while inhibition of clathrin-mediated endocytosis increased it. Inflammasome activation was also analyzed at the protein level by western blot and immunofluorescence. No significant changes were observed in the expression of proteins NLRP3, AIM2 and ASC which are involved in the formation of inflammasome, nor in their intracellular localization, regardless of the inhibition of clathrin-mediated endocytosis. Active forms of caspase-1 and pro-inflammatory interleukins IL-1β i IL-18 were also not detected. Based on this, it can be concluded that infection with HAdV-D26 does not activate inflammasome in human epithelial cells. However, infection with HAdV-D26 and inhibition of clathrin-mediated endocytosis stimulated the release of cathepsin B from lysosomes, which could be the signal for inflammasome assembly and activation.