Differential expression of the proteasome alpha6 (prosalpha6) was previously reported between Plutella xylostella strains that are resistant or susceptible to the pesticide deltamethrin (DM). This finding indicated that the prosalpha6 may be involved in DM resistance. In this article, qPCR analysis revealed that the prosalpha6 was also significantly upregulated in Drosophila Kc cells treated with DM. To better understand the contribution of prosalpha6 in DM resistance, RNA interference, heterologous expression, and a proteasome inhibitor (MG-132) were used. MG-132 was used to suppress proteasomal activity, and the dsRNA was designed to block the function of prosalpha6. The results indicated that both MG-132 and prosalpha6 knockdown decreased the cellular viability following DM treatment. Prosalpha6 was cloned and transfected into Drosophila Kc cells. The result showed that overexpression of prosalpha6 in Drosophila Kc cells conferred some protection against DM. Taken together, our results indicate that prosalpha6 is involved in Drosophila cells DM resistance.