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Topical 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) is effective for treatment of human oral precancerous lesions. This animal study aimed to assess whether topical methotrexate (MTX) pretreatment could enhance the therapeutic effect of topical ALA-PDT on hamster buccal pouch precancerous lesions. Methods: Twenty hamster buccal pouch precancerous lesions were treated with either topical ALA-PDT with topical MTX pretreatment (topical MTX-ALA-PDT group, n = 10) or topical ALA-PDT alone (topical ALA-PDT group, n = 10). The intracellular protoporphyrin IX (PpIX) level in another 12 precancerous lesions (n = 6 for either the topical MTX-ALA or topical ALA group) was monitored by fluorescence spectroscopy. Results: The intracellular PpIX reached its peak level in precancerous lesions 6.5 hours and 2.5 hours after topical ALA application for the topical MTX-ALA group (5.63-fold higher in the lesion than in the normal mucosa) and topical ALA group (2.42-fold higher in the lesion than in the normal mucosa), respectively. The complete response rate of precancerous lesions was 80% for the topical MTX-ALA-PDT group and 70% for the topical ALA-PDT group. In addition, the topical MTX-ALA-PDT group required a significantly lower mean treatment number (2.1 ± 0.6) to achieve complete response than the topical ALA-PDT group (4.4 ± 1.3, p < 0.001)). Moreover, the topical MTX-ALA-PDT group had a lower recurrence rate (12.5%) than the topical ALA-PDT group (28.6%). Conclusion: We conclude that topical MTX-pretreatment can increase intracellular PpIX production in hamster buccal pouch precancerous lesions and significantly improves the outcomes of the precancerous lesions treated with topical ALA-PDT.