Minimally ablative fractional laser devices have gained acceptance as a preferred method for skin resurfacing. Notable improvements in facial rhytides, photodamage, acne scarring, and skin laxity have been reported. The aim of the present work was to compare how different CO2 laser fluences, by modulating the secretory pathway of cytokines, are able to influence the wound-healing process, and how these fluences are associated with different clinical results. Eighteen patients, all with photodamaged skin, were treated using a fractional CO2 laser (SmartXide DOT, Deka M.E.L.A., Florence, Italy) with varying laser fluences (2.07, 2.77, and 4.15 J/cm2). An immunocytochemical study was performed at defined end points in order to obtain information about specific cytokines of the microenvironment before and after treatment. The secretory pathway of cytokines changed depending on the re-epithelization and the different laser fluences. Different but significant improvements in wrinkles, skin texture, and hyperpigmentation were definitely obtained when using 2.07, 2.77, and 4.15 J/cm2, indicating fractional CO2 laser as a valuable tool in photorejuvenation with good clinical results, rapid downtime, and an excellent safety profile.