The lytic cold-active phage HU1, a member of the family Podoviridae, infects Pseudomonas lactis and was first isolated from raw cow's milk. In this study, we used deep sequencing to determine and analyze the DNA genome sequence of HU1. We identified a 42,551-base-pair genome comprising double-stranded DNA, with 69 predicted open reading frames and a GC content of 56.4%. A whole-genome comparison did not identify HU1 as a member of any previously reported cluster of Pseudomonas phages. By contrast, HU1 was most similar to AF, which infects P. putida, with nucleotide sequence alignment coverage of 24%. These results suggest that HU1 is a novel Pseudomonas phage.