This study was conducted to simulate salpingitis of laying hens by observing the morphology and expression of inflammatory genes in the oviduct. A total of one hundred twenty 81-wk-old Roman Pink laying hens in good physical condition without the oviduct disease with an average egg production rate of 76% were fed a basal diet for 2 wks and then randomly allocated into 4 groups (6 replicates/group, 5 birds/replicate). The experimental treatments were as follows: 1) Control group (treated with PBS); 2) Organic chemical reagent (OCR) group; 3) Lipopolysaccharide (LPS) group; 4) LPS + OCR group. First, the chickens were kept upside down to make ectropion and exposure of the apertura uterinae; then prepared reagents were poured into the uterine part of the fallopian tube by using the chicken vas deferens (1 mL/layer); finally, the chickens were kept in the inverted position for 5 to 10 min. The fallopian tube samples (the magnum, isthmus, and uterus) were collected after 48 h of treatment. Compared with the control, treatment with LPS+OCR decreased (P < 0.05) the secondary villus length and primary villus area in magnum and villus length in isthmus (P < 0.05). An increase (P < 0.05) of the intervillous space of uterus was observed in LPS + OCR group compared with the control. The expressions of interleukin-6 mRNA of magnum and interferon-gamma (IFN-gamma) of isthmus in the LPS and LPS+OCR treatments were higher (P < 0.05) than that in control. Compared with the control, treatment with LPS+OCR increased (P < 0.05) the expressions of IFN-gamma mRNA of magnum and IFN-gamma, tumor necrosis factor-alpha and inducible nitric oxide synthase mRNA of uterus in laying hens. In conclusion, the results of morphological damage of fallopian tube tissue and increased expression of inflammatory factors in LPS + OCR treatment group suggested that LPS+OCR treatment can provide data basis to establish salpingitis model in laying hens for studying the pathogenesis of it.