Objectives: This study investigated the efficacy of theobromine in comparison with 0.05% sodium fluoride solution for remineralization of initial enamel caries lesions (IECLs). Materials and Methods: Ninety non-carious extracted premolars were sectioned longitudinally into buccal and lingual segments. Caries-like lesions were induced in each segment using acidified gel. Forty-five buccal segments were used for surface microhardness (SMH) test, and 45 buccal segments were used for energy-dispersive X-ray spectroscopy (EDS). The lingual segments were used as the control group for EDS and SMH test. The baseline SMH was measured with a Vickers hardness tester, and the baseline calcium content was analyzed by EDS. Each test group was divided into three subgroups for treatment with (1) artificial saliva, (2) 1.1 mol/L theobromine, and (3) 0.05% sodium fluoride. Remineralization and demineralization were done by daily pH cycling to simulate the oral environment. Samples in each group were immersed in treatment solutions for 1 min before and after the remineralizing cycle. After a 7-day cycle, the SMH test and EDS analysis were performed again. Data were analyzed using one-way ANOVA, Tukey's post-hoc test, and paired sample t-test (P<0.05). Results: All treatments effectively remineralized the IECLs (P<0.05), and theobromine caused the maximum increase in SMH, which was significantly higher than the value in sodium fluoride group (mean value of 36.56±4.95 versus 23.25±3.92; P=0.000). EDS showed the highest calcium deposition in theobromine group (3.82±1.83wt%). Conclusion: Theobromine is an effective cariostatic agent, and can be considered as a safe alternative to fluoride in preventive dental care.