Epidermal stem cell function is regulated by external cues from the environment and replenishing the epidermal cell population is initiated in the basale layer of its stratified epithelium. Like the epidermis, ruminal stratified epithelium responds to external stimuli, such as diet. The objectives of this study were to assess the effects of 2 different feeding programs on abundance of potential stem and progenitor cell populations and proliferation of rumen epidermal cells in preweaned calves. Neonatal Holstein bull calves (n = 12) were individually housed and randomly assigned to one of 2 diets. Diets were isoenergetic (3.87 ± 0.06 MCal of ME/d) and isonitrogenous (0.17 ± 0.003 kg/d of apparent digestible protein). Diets were: milk replacer only (MRO; n = 6), or milk replacer with starter (MRS; n = 6). Milk replacer was 22% CP, 21.5% fat (DM basis) while the textured calf starter was 21.5% CP (DM basis). Water was available ad libitum. Feed and water intake were recorded daily. Putative stem and progenitor cells were labeled via administration of a thymidine analog (5-bromo-2′deoxyuridine; BrdU; 5 mg/kg BW in sterile saline) for 5 consecutive days and allowed a 25-d washout period. Calves were euthanized on d 43 ± 1. Rumen tissue was obtained from the ventral sac and processed for fluorescent immunohistochemical analyses of BrdU (putative stem and progenitor cells) and Ki67 (cell proliferation). Data were analyzed using the MIXED procedure in SAS 9.4. Body growth did not differ between treatments, but empty reticulo-rumens were heavier in MRS calves (MRS: 0.67 ± 0.04 kg; MRO: 0.39 ± 0.04 kg; P = 0.001). The percentage of label-retaining BrdU basale cells was higher in MRO compared with MRS calves (2.0 ± 0.3% vs 0.3 ± 0.2%, respectively; P = 0.001). A higher percentage of basale cells undergoing proliferation was observed in MRS than MRO calves (18.4 ± 2.6% vs 10.8 ± 2.8%, respectively; P = 0.079). These results indicate that presumptive stem and progenitor cells within the rumen epidermis are identifiable by their ability to retain labeled DNA long-term and change proliferative status in response to diet.