Flowback and produced water (FPW) is an end-product of the hydraulic fracturing method of oil and gas extraction that is highly enriched in alkaline earth metals such as strontium (Sr). While Sr concentrations in FPW can exceed toxic thresholds for fish, the accompanying high concentrations of calcium (Ca) in FPW may ameliorate any toxicity. In this study, Sr bioaccumulation and molecular, biochemical, and physiological changes in ionoregulatory endpoints were investigated in rainbow trout (Oncorhynchus mykiss). Exposures were conducted over a 96-h period at Sr concentrations ranging from 1.7 to 1948 µM, with effects at the highest Sr exposure concentration also separately examined in waters of varying Ca concentration (10 to 958 µM). Plasma and gill Sr burdens increased as a function of increasing waterborne Sr, and accumulation increased further as water Ca concentrations were lowered. Despite this, there was no consistent, dose-dependent effect of Sr on plasma or gill Ca concentrations, although impacts on plasma and branchial sodium (Na) concentrations were observed. Waterborne Sr significantly inhibited branchial Ca2+-ATPase activity, albeit only at the highest tested Sr concentration (1948 µM). In exposure treatments where Sr was highly elevated and water Ca was reduced, the hepatic gene expression of Ca signaling receptors β-2 adrenergic receptor (Adrb2) and inositol-1,4,5-triphosphate receptor-2 (Itpr2) were inhibited, highlighting novel potential pathways of Sr toxicity in rainbow trout. Overall, these data indicate that water Ca has a strong effect on Sr bioavailability, but over an acute exposure period there is limited evidence for an effect of Sr on Ca homeostasis. Although Sr is elevated in effluents associated with the oil and gas industry, the co-occurrence of high Ca concentrations might protect freshwater fish against acute effects related to Sr exposure.