Endometriosis is characterized by the presence of inflamed and fibrotic endometrial tissue outside the uterine cavity. We have previously found that serpin A1 (SERPINA1) was decreased in the endometriosis-like lesion of the mouse model, which makes us hypothesize that a decrease in SERPINA1 may exacerbate inflammation in the lesion. However, the molecular mechanisms by which SERPINA1 affects the expression of inflammatory cytokines and the development of endometriosis remains unclear. To investigate the role of SERPINA1 in endometrial stromal cells (ESCs), RNA-seq analysis was performed using RNA extracted from ESCs knocking down SERPINA1. The analysis has identified several toll-like receptor (TLR)-related factors as the upregulated genes. Silencing SERPINA1 expression increased the expression of TLR3, TLR4, their downstream factor MYD88, and inflammatory cytokines in cultured ESCs. Treatment with TLR3 or TLR4 agonists enhanced the expression of inflammatory cytokines, whereas inhibitors of TLR3 or TLR4 decreased the expression of these cytokines in SERPINA1-silenced ESCs. Immunohistochemical analysis showed that TLR3, TLR4, and MYD88 were localized in the endometriotic lesion. Thus, our data indicate that the decrease in SERPINA1 induces the expression of inflammatory cytokines in ESCs, accompanying with TLR3/4 signaling. The regulation of intracellular SERPINA1 could be a potential strategy to inhibit inflammatory responses in endometriotic lesions.