Various adverse conditions can trigger defensive mechanisms in Listeria monocytogenes that can increase the virulence of surviving cells. The objective of this study was to evaluate the expression of one stress-response (sigB) and three virulence (plcA, hly, and iap) genes in L. monocytogenes exposed to a sub lethal dose of E-beam irradiation in dry-cured ham. To accomplish this, dry-cured ham slices (10 g) were immersed in a 109 CFU/mL suspension of L. monocytogenes strain S4-2 and subsequently irradiated with 1, 2, or 3 kGy. After irradiation, samples were stored at 7 °C or 15 °C for 30 days. Absolute gene expression levels were determined by RT-qPCR, and numbers of surviving Listeria cells were assessed by microbial counts after different storage times (0, 7, 15, and 30 days). At 7 °C, after E-beam treatment at doses of 2 or 3 kGy, Listeria gene expression significantly increased (p ≤ 0.05) up to day 15. Listeria counts decreased with increasing dosage. The relationship between absolute gene expression and the number of surviving Listeria cells could indicate that sublethal doses of E-beam irradiation can increase expression of the genes studied. We observed no significant influence of storage time or temperature on gene expression (p > 0.05). Listeria that survives E-beam treatment may display increased virulence, constituting a significant potential public health risk.