The strontium-to-calcium ratio (Sr/Ca) in aragonitic shells and statoliths often depends on temperature, and has been investigated for its potential availability as a sclerochronological record of the temperatures experienced during the life of the animal. Only a few cephalopod species have been subjected to rearing experiments to validate the temperature dependence of statolith Sr/Ca despite the strong demand for estimating their historical ecology, particularly of migratory squid populations. In this context, we examined the relationship between temperature and statolith Sr/Ca in the spear squid, Heterololigo bleekeri, by maintaining wild-caught immature individuals at one of three constant temperatures: 12, 14 or 16°C. A portion of statolith precipitated during the experiment was optically identified by daily increment analysis, and then subjected to Sr/Ca measurement using an electron probe micro-analyser. Regression analysis (N = 29) demonstrated the dependence of statolith Sr/Ca on temperature and the relationship was estimated as an equation Y = 9.93 (±0.29) − 0.11 (±0.02) X. However, ~90% of the total residual variance was accounted for by the among-individual variation of statolith Sr/Ca within each temperature group. Consequently, the 95% confidence interval ranged over ± 7.85°C when the temperature was estimated by inserting a Sr/Ca value into this equation. The statolith Sr/Ca values are unlikely to provide reliable estimates for absolute temperatures, but it may allow reconstruction of a time-series of relative temperatures experienced by a particular individual.