In bacteria, the enzyme catalyzing the transformation of 17-estradiol is considered the key enzyme for its metabolism, whose enzymatic activity and regulatory network influence the biodegradation efficiency of this typical estrogen. In this work, a novel 17-hydroxysteroid dehydrogenase (17-HSD) was characterized from the estrogen-degrading strain Pseudomonas putida SJTE-1, and two regulators were identified. This 17-HSD, a member of the short-chain dehydrogenase/reductase (SDR) superfamily, could be induced by 17-estradiol and catalyzed the oxidization reaction at the C-17 site of 17-estradiol efficiently. Its K-m value was 0.068mM, and its V-max value was 56.26mol/min/mg; over 98% of 17-estradiol was oxidized into estrone in 5min, indicating higher efficiency than other reported bacterial 17-HSDs. Furthermore, two genes (crgA and oxyR) adjacent to 17-hsd were studied which encoded the potential CrgA and OxyR regulators. Overexpression of crgA could enhance the transcription of 17-hsd, while that of oxyR resulted in the opposite effect. They could bind to the specific and different sites in the promoter region of 17-hsd gene directly, and binding of OxyR could be released by 17-estradiol. OxyR repressed the expression of 17-hsd by its specific binding to the conserved motif of GATA-N-9-TATC, while CrgA activated the expression of this gene through its binding to the motif of T-N-11-A. Therefore, this 17-HSD transformed 17-estradiol efficiently and the two regulators regulated its expression directly. This work could promote the study of the enzymatic mechanism and regulatory network of the estrogen biodegradation pathway in bacteria.