Dual RNA-seq has emerged as a genome-wide expression profiling technique, simultaneously measuring RNA transcript levels in a given host and its pathogen during an infection. Recently, the method was transferred from cell culture to in vivo models of bacterial infections; however, specific host cell-type resolution has not yet been achieved. Here we present a detailed protocol that describes the application of Dual RNA-seq to murine colonocytes isolated from mice infected with the enteric pathogen Salmonella Typhimurium. At day 5 after oral infection, the mice were humanely euthanized, their colons extracted, and colonocytes isolated and fixed. Upon antibody staining of cell type-specific surface markers, the fraction of Salmonella-invaded colonocytes was collected by fluorescence-activated cell sorting based on a fluorescent signal emitted by the internalized bacteria. Total RNA was extracted from cells enriched by this method, and ribosomal transcripts from host and microbial cells were removed prior to cDNA synthesis and library generation. We compared different protocols for library preparation and discuss their respective advantages and caveats when applied to minute RNA amounts that constitute an inherent challenge for in vivo transcriptomics. Our results introduce an ultralow input protocol that holds promise for cell type-specific in vivo Dual RNA-seq for charting gene expression of a bacterial pathogen within its respective in vivo niche, along with the consequent host response.