Endo-1,4-β-glucanases (EGases) are involved in editing of plant cell walls and play important roles in tissue elongation, fruit ripening, and abscission. In this study, we cloned an EGases gene PnCel1 from Panax notoginseng by RT-PCR, and analyzed its expression and function. The results showed that: (1) Exogenous methyl jasmonate, salicylic acid, gibberellin, abscisic acid, and ethephon treatment significantly induced the expression level of PnCel1, while the infection of root rot fungi including Fusarium solani and Fusarium oxysporum as well as Alternaria alternata and Fusarium equiseti significantly inhibited its expression level in P. notoginseng. (2) Subcellular localization result showed that PnCel1-GFP fusion protein was localized in the cell wall of onion epidermal cells. (3) The promoter sequence ［(-1)-(-828) bp］ of PnCel1 was cloned through chromosome walking, and a plant expression vector (pBI121-PPnCel1-GUS) of β-glucuronidase driven by PnCel1 promoter was successfully constructed, which was then transferred into Nicotiana tabacum L., and 7 positive transgenic tobacco plants were identified through PCR screening. (4) GUS activity detection indicated that 5 plant hormones can induce promoter activity of PnCel1, including methyl jasmonate, but the transcriptional activity of PnCel1 promoter decreased after infection with 4 pathogens, such as F. solani. And the PnCel1 promoter was also negatively regulated by the P. notoginseng WRKY transcription factor 5/9/12/15/27. (5) Compared with wild-type tobacco, PnCel1-overexpressing transgenic tobacco increased susceptibility to the root rot and decreased lignin content, indicating that PnCel1 may be involved in altering the cell wall structure. The above results indicate that plant hormones can up-regulate the expression of PnCel1 in P. notoginseng roots, while pathogens infection reduces the expression level of PnCel1 and inhibits the PPnCel1 activity. It is speculated that PnCel1 may increase the susceptibility of P. notoginseng to root rot by modification of cell wall structure.