Zinc is an essential trace element critical for animal growth, immune function, and antioxidant defense. Organic zinc sources chelated with amino acids are increasingly used as alternatives to inorganic zinc due to their potentially higher bioavailability. This study compared intracellular accumulation and transcellular transport of inorganic zinc sulfate (ZnS) and three organic zinc sources: zinc hydroxymethionine (ZnM), zinc valine, and zinc glycine (ZnG) in the porcine intestinal epithelial IPEC-J2 cell model, with particular emphasis on the effects of high-calcium levels. Results showed that ZnM and ZnG exhibited significantly greater intracellular zinc accumulation at 50–100 μM and higher transcellular transport efficiency than ZnS (P < 0.05), with ZnG displaying the highest transport rate (P < 0.05). High calcium (2400 μM) promoted apical zinc uptake and intracellular retention across all sources but inhibited early basolateral efflux; organic sources were less affected overall. Transcriptome analysis revealed 300 differentially expressed genes uniquely shared by ZnM and ZnG, enriched in riboflavin metabolism, fatty acid metabolism, taurine metabolism, and tight junction pathways. Under high calcium, organic zinc-specific pathways included sulfur metabolism and thyroid hormone signaling, whereas calcium broadly activated MAPK signaling and endoplasmic reticulum protein processing. These findings indicate that selected organic zinc sources offer superior transport efficiency and reduced susceptibility to high-calcium interference, likely through differential metabolic and transporter regulation.