During recent years a wealth of studies has focused on the role of polyamines in plant–environment stress interaction. However, less is known about the role of these compounds in plant disease resistance, and most of this knowledge is related to the hypersensitive response. Here the involvement of polyamines in the oat–crown rust interaction is examined, focusing on the mesophyll cell pre‐penetration and penetration resistance mechanisms. Two different oat cultivars were used, one resistant (Saia) and the other susceptible (Araceli) to crown rust (Puccinia coronata f. sp. avenae). Differential increases in polyamines between oat cultivars upon rust inoculation were observed and associated with increased pre‐penetration and penetration resistance. Elevated levels of spermidine and spermine, together with increases in the cell wall‐bound diamine‐ and polyamine oxidase activities, and concomitant increases in the polyamine oxidation product 1,3‐diaminopropane, were observed in the resistant cultivar at early stages of the infection process. This suggests an involvement of polyamines and derived H2O2 during penetration resistance. The putative role of polyamines in oat resistance to crown rust is supported by the increased resistance observed in the susceptible cultivar Araceli, as a result of reduced appressorium formation, after treatment with exogenous polyamines. Because arginine decarboxylase (ADC) has been reported as a key polyamine biosynthesis enzyme in plants exposed to stress, ADC transcript levels and enzyme activity were also evaluated, revealing an increase in ADC activity in cv. Saia at the key time points for the pre‐penetration and penetration resistance responses, which suggest an involvement of this enzyme in the resistance response.