We previously reported that cardiotonic steroids stimulate collagen synthesis by cardiac fibroblasts in a process that involves signaling through the Na/K-ATPase pathway. In this study, we examined the effect of cardiotonic steroids on dermal fibroblasts collagen synthesis and on wound healing. Increased collagen expression by human dermal fibroblasts was noted in response to the cardiotonic steroid, marinobufagenin, in a dose and time dependent-fashion. An eight fold increase in collagen synthesis was noted when cells were exposed to 10 nM marinobufagenin for 24 hours (p<0.01). Similar increases in proline incorporation were seen following treatment with digoxin, ouabain and marinobufagenin (10 nM X 24 hours, all results p< 0.01 vs. control). The co-administration of the Src inhibitor, PP2, or N-Acetyl Cysteine completely prevented collagen stimulation by marinobufagenin. Next, we examined the effect of digoxin, ouabain and marinobufagenin on the rate of wound closure in an in vitro model where human dermal fibroblasts cultures were wounded with a pipette tip and monitored by digital microscopy. Finally, we administered digoxin in an in vivo wound healing model. Olive oil was chosen as the digoxin carrier because of a favorable partition coefficient observed for labeled digoxin with saline. This application significantly accelerated in vivo wound healing in rats wounded with an 8 mm biopsy cut. Increased collagen accumulation was noted 9 days after wounding (both p< 0.01). The data suggest that cardiotonic steroids induce increases in collagen synthesis by dermal fibroblasts, as could potentially be exploited to accelerate wound healing.