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Aims:  To determine the effect of UV radiation on the viability of two strains of Mycobacterium avium ssp. paratuberculosis (Map) inoculated into milk. Methods and Results:  Mycobacterium avium ssp. paratuberculosis in a ultra heat treated milk matrix was subjected to increasing doses of UV‐C radiation from 0 to 1836 mJ ml−1 using a pilot‐scale UV reactor (20 l capacity). Survival of Map was monitored by culture on Herrold’s egg yolk medium, Middlebrook 7H10 medium and the FASTPlaqueTB™ phage assay. Differences in sensitivity to UV treatment were observed between strains, however, at 1000 mJ ml−1 a Map kill rate of 0·1–0·6 log10 was achieved regardless of strain used or method employed to enumerate Map. Although the inactivation trend was similar on the culture and phage assay, the former gave a consistently higher viable count. Conclusions:  The use of UV radiation alone does not represent an alternative to current pasteurization regimes for a large reduction in viable Map in milk. Significance and Impact of the Study:  To the authors’ knowledge the work here represents the first pilot‐scale UV treatment process used to assess UV efficacy to inactivate Map in milk. The results are similar to those obtained with a laboratory‐scale process indicating the difficulties associated with UV treatment of an opaque liquid and the recalcitrance of Map towards inimical treatments.