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Melanization, an important defense response, plays a vital role in arthropod immunity. It is mediated by serine proteases (SPs) that convert the inactive prophenoloxidase (PPO) to active phenoloxidase (PO) and is tightly regulated by serine protease inhibitors (serpins) which belong to a well distributed superfamily in invertebrates, participating in immune mechanisms and other important physiological processes. Here, we investigated the <i>Bmserpin2</i> gene which was identified from a transcriptome database in response to <i>Bombyx mori</i> nucleopolyhedrovirus (BmNPV) infection. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that <i>Bmserpin2</i> was expressed in all tissues, with maximum expression in fat body. Upon BmNPV infection, the expression of <i>Bmserpin2</i> was up-regulated in P50 (susceptible strain) and BC9 (resistant strain) in haemocytes, fat body and the midgut. However, up-regulation was delayed in BC9 (48 or 72 h), in contrast to P50 (24 h), after BmNPV infection. Meanwhile, Bmserpin2 could delay or inhibit melanization in silkworm haemolymph. Significant increased PO activity can be observed in <i>Bmserpin2</i>-depleted haemolymph under NPV infection. Furthermore, the viral genomic DNA copy number was decreased in <i>Bmserpin2</i>-depleted haemolymph. We conclude that <i>Bmserpin2</i> is an inducible gene which might be involved in the regulation of PPO activation and suppressed melanization, and have a potential role in the innate immune system of <i>B. mori</i>.