Background Lactobacillus rhamnosus Pen is a human endogenous strain with well-documented health promoting properties that is used for production of probiotics. It has a long safety history of application, and its effectiveness in the prevention of antibiotic-associated diarrhoea has also been confirmed in clinical trials. Results Here we present the complete genome sequence of L. rhamnosus Pen, which consists of a circular 2,884,4966-bp chromosome with a GC content of 46.8%. Within 2907 open reading frames (ORFs), genes involved with probiotic properties were identified. A CRISPR locus, consisting of a 1092-nt region with 16 spacers, was also detected. Finally, an intact prophage of ~ 40.7 kb, 57 ORFs, GC content 44.8% was identified. Conclusions Genomic analysis confirmed the probiotic properties of L. rhamnosus Pen and may indicate new biotechnological applications of this industrially important strain.