Yeast Atg8 and mammalian microtubule‐associated protein light chain 3 (LC3) are landmark proteins essential for autophagy. Here the lepidopteran Atg8, a homolog of LC3, is characterized. Sequence analysis reveals that Atg8 proteins are highly conserved in lepidopteran species. The abundance of endogeous Atg8 and the ratios of Atg8 conjugation to phosphatidylethanolamine (Atg8‐PE)/Atg8 are different among several lepidopteran cell lines and different tissues of Helicoverpa armigera larvae. Both the density of fluorescent pre‐autophagosomal structures with GFP‐Ha Atg8 and the abundance of Atg6 are positively correlated with levels of Atg8‐PE in different cell lines. The mutant GFP‐Atg8G116A has lost the function in punctual formation, suggesting that G116 is important for autophagy.Exogenous factors have significant influences on the conversion of Atg8 in lepidopteran cells. Bacillus thuringiensis enhances the degradation of Atg8 in Spodoptera litura Sl‐HP cells. Atg8‐PE degrades gradually with extension of amino acid starvation, and bafilomycin A1 can block the decrease through the inhibition of autophagosome fusion with lysosome. Interestingly, high pH is more effective than amino acid starvation in Bombyx mori Bme cells to induce the conversion of BmAtg8 to BmAgt8‐PE. Change of the quality of fetal bovine serum in the culture medium results in alteration of the ratio of Atg8‐PE/Atg8 in some lepidopteran cell lines.