Mutations of cilia expressed proteins are associated with an attenuated shear-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]i) in renal epithelial cell lines derived from murine models of autosomal recessive PKD (ARPKD). We hypothesized that human ARPKD cyst-lining renal epithelial cells also exhibited dysregulated mechanosensation. To test this, conditionally immortalized cell lines derived from human fetal ARPKD cyst-lining (pool and clone 5E) cell lines with low levels of fibrocystin/polyductin expression and age-matched normal collecting tubule (HFCT pool and clone 2C) cell lines were grown in culture, loaded with a Ca²⁺ indicator dye, and subjected to laminar shear. Clonal cell lines were derived from single cells present in pools of cells from cyst-lining and collecting tubules, microdissected from human kidney. Resting and peak [Ca²⁺]i were similar between ARPKD 5E and pool, and HFCT 2C and pool; however, the flow-induced peak [Ca²⁺]i was greater in ARPKD 5E (700±87 nM, n=21) than in HFCT 2C (315±58 nM, n=12; p <0.01) cells. ARPKD 5E cells treated with Gd³⁺, an inhibitor of non-selective cation channels, inhibited but did not abolish the shear-induced [Ca²⁺]i transient. Cilia were ~20% shorter in ARPKD than HFCT cells, but no difference in ciliary localization or total cellular expression of polycystin-2, a mechanosenory Gd³⁺-sensitive cation channel, was detected between ARPKD and HFCT cells. The intracellular Ca²⁺ stores were similar between cells. In sum, human ARPKD cells exhibit an exaggerated Gd³⁺-sensitive mechano-induced Ca²⁺ response compared to controls; whether this represents dysregulated polycystin-2 activity in ARPKD cells remains to be explored.