The inability to propagate porcine bocavirus (PBoV) in vitro has severely impeded research into its fundamental biology and pathogenic potential since its discovery 15 years ago. This study reports the successful isolation and characterization of a novel PBoV strain, PBoV-CNH, from diarrheic piglets in China. Crucially, PBoV-CNH was isolated and propagated in LLC-PK1 cells, a kidney-derived cell line from 3-4-week-old pigs, matching the age of the susceptible host. This represents the first documented isolation of PBoV in a continuous cell line. The isolate exhibited typical bocavirus morphology (20-30 nm particles), shared 94.15% whole-genome nucleotide identity with the NCBI reference strain (NC_016031.1), and displayed hemagglutination activity (HA) characteristic of Parvoviridae. Phylogenetic analysis revealed that PBoV-CNH clusters within a clade containing human bocaviruses (HBoVs), highlighting close genetic relatedness. Experimental infection of piglets confirmed PBoV-CNH as a primary pathogen. Notably, the virus exhibited dual tissue tropism: orally infected piglets developed acute diarrhea with high intestinal viral loads, while intranasally infected piglets showed diarrhea, significant lung pathology, and the highest viral loads in respiratory tissues. This efficient replication and shedding in the respiratory tract, combined with phylogenetic proximity to HBoVs and a previously reported human-PBoV infection case, signals a tangible risk of cross-species transmission. To our knowledge, this work constitutes the first successful in vitro propagation of PBoV and provides definitive experimental evidence, fulfilling Koch's postulates, of its in vivo pathogenicity and tissue tropism. These findings provide essential tools and foundational insights for future research into PBoV biology, transmission, and control strategies.