Nosema ceranae is a widely prevalent microsporidian parasite in the western honey bee. There is considerable uncertainty regarding infection dynamics of this important pathogen in honey bee colonies. Understanding the infection dynamics at the colony level may aid in development of a reliable sampling protocol for N. ceranae diagnosis, and provide insights into efficient treatment strategies. The primary objective of this study was to characterize the prevalence (proportion of the sampled bees found infected) and intensity (number of spores per bee) of N. ceranae infection in bees from various age cohorts in a colony. We examined N. ceranae infection in both overwintered colonies that were naturally infected with N. ceranae and in quadruple cohort nucleus colonies that were established and artificially inoculated with N. ceranae. We also examined and quantified effects of N. ceranae infection on hypopharyngeal gland protein content and gut pH. There was no correlation between the prevalence and intensity of N. ceranae infection in composite samples (pooled bee samples used for analysis). Our results indicated that the prevalence and intensity of N. ceranae infection is significantly influenced by honey bee age. The N. ceranae infection prevalence values from composite samples of background bees (unmarked bees collected from four different locations in a colony) were not significantly different from those pertaining to marked-bee age cohorts specific to each sampling date. The foraging-aged bees had a higher prevalence of N. ceranae infection when compared to nurse-aged bees. N. ceranae did not have a significant effect on hypopharyngeal gland protein content. Further, there was no significant difference in mean gut pH of N. ceranae infected bees and non-infected bees. This study provides comprehensive insights into N. ceranae infection dynamics at the colony level, and also demonstrates the effects of N. ceranae infection on hypopharyngeal gland protein content and midgut pH.